BIOL 220 Microbiology- Nurses





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Module 2 Module 18 Module 36

Module 3 Module 19 Module 37

Module 4 Module 21 Module 42

Module 5 Module 22 Module 43

Module 6 Module 23 Module 45

Module 7 Module 27 Module 46

Module 8 Module 28 Module 47

Module 9 Module 29 Module 49

Module 10 Module 30 Module 50

Module 11 Module 31 Module 51

Module 12 Module 32 Module 52

Module 13 Module 33 Module 53

Module 15 Module 34 Module 54

Module 16 Module 35 Module 55

Module 17 Module 38

Module 20 Module 39




Preparing and Dispensing Media




This module will be done primarily as an introduction to the microbiology lab and to the techniques that are used by our microbiology technician to prepare the media that will be provided for you as needed.


However, as many of you may someday need to do these techniques yourself, it is important that we go through the basic steps. Many of you may someday go over seas on short or long-term missions trips. Others may return to your home in these countries or in some small town in America where clinical labs are not available to do identification work for you. In each of these circumstances, you may very well find yourself having to perform these basic microbiological techniques on your own.


Media is the material we use to provide the microbes with most of the requirements they need to live out side their normal habitat. It is one of the most important thing that Robert Koch had to develop in his pioneering work on the Germ Theory of Disease. While the incubator provides the correct temperature and lighting conditions, it is the media that provides the nutrients, correct pH, moisture and other essential requirements. Therefore, there are many types of media available due to the varied requirements of the different microbes.


Regardless of the specific type of media, all media comes either as liquid, usually called broth, or semi-solid, called agar. The broth is provided in test tubes or flasks while the agar may come in test tubes, as slants or deeps, or in petri plates.


Preparing the media, regardless of the type, requires the same basic procedure. After you have determined the type of media to use you must gather the ingredients together. In most modern media, the ingredients are already mixed together and you may only need to add agar. The ingredients are then weighted out according to the directions which are usually provided for making up 1 liter. If more or less than that is required, the amounts must be increased or decreased accordingly. After weighing, the dry ingredients must be mixed into the water which is in a flask that is twice the volume of the final amount. This is then placed on a heating plate with a stir bar and allowed to hear just until boiling begins.


At this point, the next step depends on whether you are making plates or tubes. If you are making plates, the agar is autoclaved in the flask and the plates are poured afterwards. If you are making anything in a test tube, the tubes are poured first, then autoclaved and allowed to cool.





1. What is the basic device used to properly measure liquids in the lab?


2. What is the name of the device used to pour the media into the test tubes?


3. The directions for most media are given for making up how much total media?





None as this is only a demo




1. Explain the steps that must be followed to correctly make up a nutrient agar slants.


2. Why do we need media in the microbiology lab?




Sterilization of Media and Equipment




Whether you are making media or changing a patients bandages, it is imperative that proper sterility is maintained at all times. As you will see in the next exercise, bacteria, fungi, viruses and other microorganisms exist all around us, on us and in us. If you are not careful, some of these can get into your media or in your patient with serious effects.


Just as maintaining aseptic conditions in the clinical setting requires a certain set of procedures, keeping media and cultures contamination free requires its own set of methodologies. In the lab setting we are usually interested in keeping media and equipment complete sterile through the process of sterilization. This means that absolutely no organisms are present. When we are involved in transferring and culturing a single microbe type, we are then primarily interested in maintaining purity.


The most common way to do this in the lab is through the use of heat. The technique known as flaming uses very hot, dry temperatures to kill microbes on metal and glass pieces of equipment. The use of a dry oven is similar. Unfortunately, dry heat will not help when we are trying to sterilize liquids, large quantities of material or clothing. Wet heat can be used for several of these items and can be applied in a variety of ways. The simple process of boiling can be used to sterilize liquids but it takes a long time and results in the loss of liquid and change in its concentration. Steam avoids some of these problems but also takes a long time as it can only reach 1000 C. Therefore, the most useful sterilizing technique available today is the use of steam under pressure, which is commonly done in an autoclave. In most modern autoclaves, the pressure is allowed to reach 15 psi and the temperature can reach 1210C before boiling occurs. This allows much faster sterilization of material, usually requiring just 15 minutes, and can be used for solids, liquids and, in some cases, material.


Unfortunately, the reason heat is so effective is also the reason it cannot be used in some instances. In many cases, the proteins, amino acids and other chemical compounds in media are themselves destroyed by heat. In that case we can sterilize the media through filtration. In this technique, liquid media is filtered through filters that have pores so small that they stop even viruses. The filtrate is collected into a sterile container and is then poured.


In many cases, such as with large batches of clothing or bedding, neither of these techniques is practical. For these types of material, sterilizing gases may be used. These are toxic gases that can be released into large sealed chambers containing the material to be sterilized. The toxic gas kills the microbes as it penetrates through the clothing, etc.


Finally, sterilization is occasionally done using radiation of different types. This can be as mild as using ultraviolet light in a restaurant to gamma radiation in sterilizing fruit.





1. Define sterilization


2. What is the size of the pores needed to properly sterilize liquid media?


3. At what temperature does water boil if it is under 25 psi?




None as this is only a demo





1. What is the relationship between the autoclave and a pressure cooker?


2. What are the proper conditions that most autoclaves are run at?


3. In what types of sterilizing techniques would moisture be a problem?





Compound Microscope for the Study of Microbes





Since we assume that you have used microscopes in previous science classes, we will spend only a short time reviewing the structure and use of a microscope. However, since these each cost about $2,000.00 and even the smallest lens costs over $100.00, please ask for assistance if you do not understand or forget any of this material.


Due to the extreme size, by definition, of microorganism, the microscope will be one of our primary tools in this course. The ones we are using are most completely called "compound binocular light microscopes". "Compound" refers to the fact that two lenses are used to magnify the objects, "binocular" refers to the two oculars present, rather than the one in regular scopes, and "light" refers to the way the images are produced.


It is imperative that you study the diagrams in the manual and become familiar with the parts of the microscope and their functions. As you do this you will notice that there are several differences between this scope and those you have used in the past that make it much easier to use. First, there are the two oculars which allow you to use both eyes without having to close one eye. To properly use these, be sure to adjust them out/in so that you get one circle of light, not two. Then use the fine focus ring on one of the oculars, which one varies from scope to scope, to adjust for the differences between your left and right eyes.


Second is the degree of control you have over the light amount. Each of these have a rheostat switch that allows you to regulate the amount of light produced by the bulb. Usually, this should be set a maximum to get the whitest light. Then there is the diaphragm that opens and closes allowing more and less light, respectively, through it. Finally, the sub-stage condensor moves up and down beneath the stage focusing a cone of light into a single point. By raising and lowering it you can change where the pinpoint of light falls, changing the amount of light on the slide.


Finally, you will notice that the stage has a rather refined mechanism for moving the slide around on it. This stage manipulator will allow precise movement of the slide in a horizontal and vertical direction allowing you to carefully search the slide or to track an organism.


Another difference between these and the less expensive scopes you may have used is the addition of a fourth objective, the 100X oil immersion lens. This lens allows greater magnification which is necessary for seeing the bacteria that are our primary goal. It is able to do this because it is designed to be used with a thin layer of oil between the slide and the lens. This oil has the same index of refraction as the glass slide and the glass lens. This way the light will not refract, or bend, as it passes from one layer to the next. This allows more light to pass through into the lens to create a better image at that high magnification.




1. How can you determine the total magnification that you are using?


2. Define parfocal.


3. How can you control the light on your scope?





As you prepare for the test, be sure that you know all the parts of the microscope and their function. Be sure that you understand the important terms and that you know how to properly use the instrument. One of the most important thing to keep in mind as you go through this course, is the relative size of the organisms you are looking at. While we will rarely be concerned with the exact size of an organism, it will be important to keep in perspective the size of a human blood cell versus the size of a paramecium versus the size of a bacteria. This will help you know exactly what to look for and when you have found the right thing.





1. Identify the parts of a scope and give their primary function.


2. What is the complete name of the type of scope we use in this course?


3. What happens to the field of view as you increase magnification?





Cleaning Microscope Slides and Preparing a Wet Mount





While we will primarily be examining prepared slides in this class, we will frequently be making our own slides. When we view bacteria we will usually use a special set of procedures that will be explained during the staining labs. However, there are times when we will be using these techniques to view living bacteria, protozoans and fungi.


The first thing to keep in mind is the necessity for a clean slide. This is usually simplified in this class as we rarely reuse slides here due to the potentially pathogenic nature of the bacteria we use. However, it is always a good idea to examine the slides carefully and clean them completely if they appear dirty. Any dirt or grit may look like a microorganism under magnification and the oil the may remain from fingerprints may cause the stains and smears to not adhere.


The primary technique we will use to examine living organisms under the microscope will be the wet mount. This is a relatively easy technique that is done by simply placing a drop of the solution containing the organism on the slide. If you are working from an agar slant, for example, you may have to first place a drop of distilled water on the slide and then, using aseptic techniques, transfer a loopful of bacteria to the drop of water. Either way, the next step is to simply place the coverslip onto the drop in such a way that you to do not trap any air bubbles. The easiest way to do this is to first put one edge of the coverslip on the slide and slide it until it just hits the water drop. At this point, releasing the other edge of the coverslip so it falls down onto the water drop at an angle will force most of the air out to one side, reducing the number of air bubbles.


Occasionally, we may find ourselves using an alternative method called the hanging drop technique for viewing these objects. This is a way to prevent the squashed affect of having the coverslip resting right on top of the slide. Instead of following the directions in the manual we will either place the Vaseline right on the slide itself and then add the water in the middle of the ring of Vaseline or we will use a depression slide that has a thicker slide with a well that will take a drop of water. Placing the coverslip on top of this well will have the same effect as using the Vaseline.


However the most important aspect of this lab is learning and beginning to recognize the three primary shapes of bacteria cells as this is a critical aspect of bacterial identification. As shown in the lab manual, there are three basic shapes: cocci (spheres), bacilli (rods) and spirilla (spirals). However, there are modifications of each that may be seen and require additional study to differentiate.




1. Name and describe the 3 basic shapes of bacteria cells.


2. Why is using the oil immersion lens difficult with a wet mount?


3. What is a good commercial cleanser for cleaning slides?





Notice that you can frequently know the shape of the organism by its name such as Bacillus and Micrococcus. Think back over the techniques you used and the success you had in not producing too many bubbles in the slide. More than one person in the class probably was embarrassed to find that the beautiful cell she or he was drawing was actually an air bubble. As you complete this lab, think over the various sizes of the organisms and place them in order from largest to smallest.





1. What are the two ways to view living bacteria?


2. How did we modify the hanging drop method?


3. What was the hardest part of using the microscope?




Ubiquity of Microorganisms




This is a fun lab that is also designed to teach a very important lesson that is critical to your success both as a microbiologist in the lab and as a medical professional: microorganisms are all around you!! They are on you, in you, floating in the air, on the table top and EVERYWHERE!! Thus, if you are not careful, you may end up with an unwanted microorganism growing on your petri plate or, more importantly, on the catheter you just inserted into your patient.


This will also be your first exposure to what a group of microorganisms growing together on agar look like. This colony is an important concept in microbiology and is assumed to represent one original cell that, over the incubation time, reproduced numerous times until enough cells were present to become visible to the naked eye. Sometimes these colonies, however, run together as the result of numerous cells being clumped together or lying very closely together.


Another important concept that should come across in this lab is the effect that antimicrobial agents have on microorganisms. You will be doing before and after samplings at several points today. Be sure to remember and compare these together when you check them in a few days.





1. Define ubiquity.


2. What impact does the term have for your work in the lab?





As you examine these plates and tubes be sure to look at the lab exercise to refresh your memory of what you did to each plate or tube originally. Examine the soil tube to notice where the growth seems to occur. Most likely it will be throughout the entire tube with some on the bottom, some mixed throughout the broth and some floating on top. We will discuss what each of these growth patterns are called and what they represent in another exercise.


Be sure to look at all the other group's air plates as they will each have come from a different location. Notice that these plates probably have the most distinct colonies as the microorganisms were floating as single cells and landed in separate areas of the plate. There are also probably both fungal and bacterial colonies since both are present in the air. The fungal colonies are usually larger, fuzzier, and more likely to be colored than the bacterial colonies that are smaller, smoother or slimier, and tend to be whitish.


In two of these experiments, you made before and after cultures. Notice the difference between the number of organisms on the plate taken from the unwashed benchtop and the washed benchtop. Is there a difference in number? Does it appear that the material used to wash down the benchtop was effective against the microbes? Now think about the results from the handwashing experiment. Which had more organisms: the before or after washing? This might make you think that the soap used in the lab is ineffective. However, think about the kind of handwashing we do here in the lab versus the type of hospital or surgical scrub you use in that setting. In the lab, we are interested primarily in just getting rid of any microbes we might have picked up during lab, which would be just sitting on the skin surface. However, in the surgical setting, we are primarily concerned with anything that we might bring in as normal skin flora that might be lurking under the top layers of skin. Thus, a surgical scrub is designed to get down past those initial layers to fresh, uncontaminated skin. Our lab washing, however, just removes surface microbes and may actually expose more microbes existing as normal flora under our top layers.




1. Why should you label the petri plates before removing it from the tray?


2. How can you tell the difference between the bacteria and fungi colonies?


3. Which plate had the clearest, most separate colonies?


4. what happened to the number of colonies after you washed your hands? Why?





Aseptic Transfer of Microbes





As we just learned in exercise 7, microorganisms are everywhere. In the lab, we need to be able to work with our material, primarily sterile media, in such a way as to prevent those microorganisms from getting into or onto our media. Sometimes this will mean trying to maintain sterility, such as when we pour a plate and do not want anything to get on it initially. Other times it will mean trying to keep anything from getting on the media except what we deliberately put there ourselves.


This is what we will be learning in this lab. We will be trying to take a specific bacteria and place it onto or into sterile media so that it, and only it, is transferred from the pure stock culture to the new media. Being able to do this allows you to insure that the cultures you work with only contain the one species of organism you want it to and no contaminants that might affect your studies.


There are a few helpful hints that you need to keep in mind as you do this:


o Always assume that anytime a sterile item is exposed to air the possibility of contamination exists.


o Never open or expose a sterile item until you are ready to use it.


o Always keep the things you are working with in your hands. Do not put them down, even if it is on your cloroxed table cloth.


o Be sure to flame your inoculating needle or loop thoroughly and to let it cool for a few seconds before putting it into a culture.





1. Define aseptic.


2. Using the techniques discussed here, how many test tubes will you have to hold at one time?





You will know if you have been successful with your transfers if each slant contains growth and if that growth is clearly from only one species of microorganism. The particular organism you are using in this experiment will help you see that more easily because of its obvious pigmentation.


On a slant there should be a nice straight line or zigzag line, depending on what you did, running along the slant. There will also probably be an accumulation of liquid at the bottom of the slant but that is normal. In a broth culture, you should see only the pigmentation of the transferred bacterium and it should all be either on the top, at the bottom or evenly dispersed throughout the broth.





1. What color was the bacteria on the slant?


2. What must never happen to the test tube tops?


3. Why should you wait 15 sec after flaming before transferring microbes?





Aseptic Use of a Serological Pipette





In a science lab such as this, we are always concerned with exactness, whatever it is that we are doing. This is particularly true when we are talking about measuring out amounts of material. One of the things we will be measuring out many times this semester is liquid, whether in measuring material to add to a broth culture or actually measuring out a given amount of that broth culture to put into something else.


For this type of measuring of liquids a serological pipette is usually the simplest, safest, and most accurate way to do this. A serological pipette is a devise that looks like a long tube with a narrowed tip and markings all along its length. At the top end will be a marking such as "1 in 1/100" or "10 in 1/10". The first number always tells you the total volume that the tube is capable of holding within the gradations while the second number tells you what the smallest markings indicate. Thus the first tube measures 1 ml and can measure it in 1/100ths of a ml while the second one holds a full 10 ml but each smallest marking only measures 1/10th of a ml.


In some cases, such as the 1 ml pipettes that we use in this lab, there may also be a few markings above the "0 ml" point. This will usually allow you to measure out an additional .1 ml so that, as we will be doing later in the course, you can measure out 1.1 ml so that you can do serial dilutions of 1 ml and .1 ml at the same time.


While the manual talks about glass pipettes that are reusable and have to be cleaned and sterilized, our pipettes are usually disposable and come already sterilized. However, remember that as soon as they are removed from the package, they are no longer considered to be sterilized and should be used immediately and then disposed of properly.


When using a serological pipette there are several things to keep in mind:


o The narrowed tip end of the pipette is the most inaccurate portion of the pipette. Therefore, only use it when measuring out the full contents of the pipette.


o Anytime liquid is contained in a narrow tube it forms a meniscus due to the attraction of the walls of the tube. This can create confusion as the meniscus can form a line around the edge of the tube as well as one at the depression in the center. Always read from the bottom of the depression.


o Never orally or mouth pipette as one inadvertent "slurp" can cause you to ingest whatever it is you are pipetting. Always use the pipette pumps provided.


o When using a pipette pump, always use the correct size (green for 5 and 10 ml and blue for 1 ml), insert the pipette with a gentle twisting motion, grasping the top of the pipette, and watch the level carefully so you do not pull liquid up into the pump.


Following these simple rules will allow you to safely and successfully measure liquids accurately.




1.What is a pipette used for?


2. What do you do with an disposable pipette as soon as you are done with it?


3. What is the meniscus?





None as this is primarily a demo.




1. Why should you never orally pipette?


2. How can you distinguish between a 10ml and a 1ml pipette?


3. What tells you what the smallest markings on the pipette means?





Aseptically Dispensing Agar into Petri Dishes





In most cases in this course we will provide you with petri dishes that already contain the agar in them. Occasionally, however, we will have you do pour plates, usually during serial dilutions. When this becomes necessary, you will need to pour the plates correctly to ensure success.


The first thing that will be needed is to melt the agar in the tubes. This also may be done for you if the tubes are already in a water bath. If not, it will be necessary for you to put the tubes in a beaker, fill it with water until it just covers the media and then place the beaker over the Bunsen burners. When the tubes are melted, which can be determined by gently shaking them using a test tube holder or by looking for a clearing of the media, the tubes can be removed from the water.


Before pouring the agar, be sure that the plate is right-side up so that you are pouring into the smaller diameter, deeper bottom of the plate. If you are pouring the agar into an empty plate, simply pull off the top of the test tube and quickly lift the edge of the plate just enough to allow the test tube lip to enter the plate. Then pour the agar into the plate in one swift motion and lower the top again. Gently swirl the plate on the table top to disperse the agar throughout the plate.


If you are pouring the agar into a plate containing some pre-added broth, such as in a serial dilution, you cannot pour the agar immediately after removing it from the boiling water as it will be too hot. Therefore, first cool the media slightly in a process called tempering. This can be a tricky process as it must be warm enough not to solidify and cool enough not to harm the microbes. The best way to do this is to simply place it in a water bath at 55o C for about 10 minutes. If such a water bath is not available, simply keep an eye on the test tube by gently touching the tube every minute or two. If you can hold the tube without burning yourself but it is still warm, that is the right time to quickly pour it.


Once the hot agar hits the cool plate it will cause condensation to build up on the top of the plate. This is the reason we invert all petri dishes when we put them into the incubator as it prevents the water from falling down onto the colonies and dispersing them. This condensation can be reduced by stacking the cooling plates immediately on top of each other so that the heat from the top warm drives off some of the condensation from the plate below it.




1. Why would you want to use a petri dish rather than a slant?


2. From what do you pour the agar for the plate?





None as this is primarily a demo.




1. What might happen if you do not swirl the plate carefully and completely after pouring the agar into it?


2. What happened if you waited too long to pour the agar from the test tube?


3. a. What happens, and why, to the top of the petri dish after the hot agar is poured into it?

b. How can you avoid this happening?





Loop-Inoculated Pour Plates





In nature it is extremely rare to find microorganisms growing in perfect isolation from each other. If you remember back to module six when we tested the soil, the air, and your lips, among other things, the organisms were mixed together and not separated. However, we need to work with pure isolates of just one species so we somehow have to be able to separate or isolate out each species from a mixed group. This is particularly important in growing cultures from humans as, when you take a throat, urine, fecal, etc. sample from a patient, there will be both the pathogen and the normal body flora present. We must be able to isolate the possible pathogen from the normal microbes in order to identify it and know the exact nature of the problem.


There are basically three ways to do this. The first one is called a pour plate and is the techniques we will use today. The other two are the streak plate and the use of selective-differential media and we will use those techniques in the future. In a pour plate the idea is to place the mixed colonies in a liquid and mix them throughout the media well enough to separate the cells and dilute them throughout the media. The media is then poured into a plate and each separate cell is then allowed to grow into individual colonies. Frequently, there will be so many cells that you are still unable to separate and identify them from each other. In that case we usually do a serial dilution which uses dilution blanks of known amounts to further dilute the number of cells so that clear, separate colonies are formed.


In this experiment we will use two dilutions of the same bacteria. Instead of using a pipette to transfer an exact amount of culture we will simple use a loop as we are more concerned with technique than quantities. Notice the stress placed on thoroughly mixing the agar before pouring it. We will use a more efficient method of mixing than diagrammed here, however. Instead of moving it back and forth in our hands we will use a Vortex Mixer that cerates a much greater mixing action.


Remember also not to add the loopful of culture to agar that is too hot or the bacteria will be killed immediately. Be sure to temper the agar as discussed previously.





1. Why are bacteria added into melted media?


2. Define colony.





Notice that both plates probably had way too many colonies to count. In fact, if you did not look closely, you probably thought there was nothing growing as there were so many that each grew only a very small amount. The very small size may also be due to the fact that so many of the colonies are growing in or under the agar and are thus suffering both from the large numbers of colonies, the effort of pushing against the agar itself, and, perhaps, the lower amounts of oxygen within the agar itself.


When we do this technique again, in conjunction with a serial dilution, there will be fewer cells in each plate resulting in fewer and larger colonies. Since we assume that the mixing action separates the cells and each colony represents groups of cells al arising from the same original cell, we could thus go in and take a sample of any one colony and transfer it to a fresh agar slant to create a pure colony. Thus, we would be isolating single species from a mixed group.




1. Describe the different appearances of the colonies.


2. Which plate (from which dilution) created the most colonies?,





Quebec Colony Counter





In order to accurately and easily count and view colonies growing on agar plates, it is helpful to use a Quebec Colony Counter. This device has a number of features that make it easier to view and count colonies. First, it has a nicely backlite viewing screen. This screen is also etched with a grid that has the entire area covered in small squares and a number of those squares, forming an "X" across the screen, are further divided into smaller squares. Finally, there is a large magnifying lens that can be adjusted to enlarge the entire plate making it easier to view and count the colonies present.


The first decision that must be made in counting colonies is whether or not there are too many to count. Sometimes you have to realize that even with the Colony Counter there are simply too many colonies to keep straight which you have counted and which you have not. Then the decision has to be made as to whether or not there are few enough to count but too many to count the entire plate. In this case, only those colonies that fall into the "X" are counted. For our plates this means that you have counted about 20% of the entire plate so we multiply the number of colonies in the "X" by 5.5 to arrive at a whole plate count. Finally, if there are few enough colonies so that you can count them all, it is a simple process to start at one corner of the plate and, using the grid lines, move across the plate counting all the colonies that fall in that row. When the end of the plate is reached, simply move down one row and count colonies in the opposite direction. In either of these last two cases you must decide before hand what to do with colonies that fall on the line. It is best to simply say that you will count them in the upper row they fall into and not in the lower row they fall into.


When using this device it is best to center the plate on the screen, turn on the light, adjust the magnifying lens so that a large, clear image is seen. Sometimes it is best to count from the top of the plate, other times it is easier to count from the bottom of the plate, through the agar. This is true particularly if the condensation is thick on the cover. WHATEVER YOU DO, DO NOT REMOVE THE PETRI PLATE LID IN ORDER TO SEE THE COLONIES BETTER!! THIS WILL PROVIDE THEM WITH THE POSSIBILITY OF EITHER BEING CONTAMINATED OR CONTAMINATING THE LAB.





1. Why is a Quebec colony counter used?


2. What does the counter have that helps it do its job?





This exercise is done with the dilution pour plates from exercise 10 which means you probably were not able to count the colonies as there were too many of them in either plate. If you were, however, you should have a pretty good feel for the unit and the process. However, if you were not able to really use it you will make use of it later on in the class.




1. When do you count only what is in the "x" ?


2. When you do that, what do you multiply your count by to determine the count for the entire plate?


3. What was the count for your plates?




Streaking for Isolation





This lab teaches you the primary way to isolate pure colonies from mixed growths of microbes. The technique is called streaking and is usually used in conjunction with the third technique called selective-differential media. Today, however, we will simply concentrate on the technique of using an agar plate to dilute a loopful of culture over an entire plate so that, eventually, individual cells are dropped off the loop to grow into pure colonies that can be used to create pure cultures in new slants.


While there are many ways to do this, the method described in your manual is the most efficient and easiest to learn. The pattern of the four sections is actually drawn on the underside of the dish and labeled using roman numerals. Then, the original loopful of culture, or cotton swab of culture, is used to thoroughly inoculate the "0" section. Care must be taken to stay within the section but here you can go back and forth over your own line of inoculation. If you are working with a cotton swab, from a throat culture perhaps, you then toss the swab and continue with a flamed loop. If you are starting with a loop you simply flame it well to remove any excess bacteria. Remember the idea is to dilute the cells across the four sections so any bacteria still hanging on the loop will only add to the number of cells moved into the next section. Rotating the plate so the next section is across from you, open the plate very slightly and drag the loop once across the "0" quadrant and right into the "I" quadrant. Now you must be careful not to go outside section "I" and not to cross over the inoculating line you create in section "I". The process is then completed, while flaming the loop after each section, by dragging the loop across one section and into the next section.





1. What is the term for how bacteria divide?


2. What is the purpose of doing a streak plate?


3. Define confluent growth.





If, when you check these plates, you find massive growth without any clear distinctions on the first one, two or even three sections, that is fine. As long as in one of the quadrants there are small clearly distinct and separate colonies, you have been successful. Hopefully, this will occur in either sections two or three as this would be ideal.


If you had little or no growth on any section it may have been due to there being very few cells in the original stock. However, it may also have been due to the fact that you did not cross over the previous section's inoculating line or that the loop was too hot and the microbes were killed when you first touched them.


If you had too much growth in all sections it was probably because you did not flame after each section, you crossed over into previous sections and grabbed additional cells or, if you were really not following directions, you actually reinoculated the loop in-between every section!!


Take special note of the plates that did work and notice the technique you used in order to insure that you can repeat this important technique successfully each time you use it!




1. How did you know if your streak was successful?


2. In which quadrant did most of your single colonies appear?


3. If you had a hard time getting a single colony what might have gone wrong?


4. What does the presence of a colony tell you?





Cultural Characteristics of Bacteria





Now that you have an idea of what a colony is, what it looks like and how to get nice separate, distinct ones from mixed cultures, we need to start working on learning the special characteristics that distinguish one species' colony from another species colonies. In Module 6 we already talked about the differences between most fungi colonies and most bacterial colonies. Now we will look more closely at the differences between different types of bacterial colonies.


Here, as throughout most of the rest of the class, we are not interested primarily in memorizing the characteristics of each species we use. Those kinds of things can be looked up in a text book should you ever need it someday. Rather, we want to concentrate on the various aspects of a colony that can be used to distinguish them from each other. That way, should you ever have to identity species based on colony characteristics, you can look up the species in Bergey's Manual of Determinative Bacteriology and understand what is meant by the description that is offered there. In this activity we will concentrate on the appearance of colonies on agar plates and in nutrient broth. However, additional details could also be discussed to describe streaks on slants.


On agar plates, colony size and shape are two of the primary aspects we are interested in . Be sure to learn the ones given in the book. In addition, the appearance of the edge of the colony, also called its margin, is also important. Finally, color is something that is also important as there are many variations of pigmentation that bacteria produce. However, the one aspect that the manual mentions that we will not worry about is the elevation as that is very difficulty to ascertain as this point in your microbiological career!


As you will see in this experiment, these characteristics may vary due to temperature, pH, or the presence of certain other environmental factors. In another module we will see that color changes with temperature as the proteins are affected by the heat. Here we will look at the difference between growth on different types of agar and the affect, for example, that salt has on some organisms.


Finally, we will frequently be growing cultures in broths and many species have a distinct type of growth when growing in liquids like that. Usually, this is due to their need for a certain type of oxygen requirements, which we will discuss further in future labs. If an organism ends up growing primarily on the top of the broth, in a thick layer, it is called pellicle growth. If it likes to grow throughout the broth, so the whole test tube is cloudy, it is said to exhibit turbid growth. Finally, many organisms will grow on the very bottom of the test tube. Your manual mentions only one such growth pattern, sediment. This is the type of bottom growth that simply rests on the bottom and is easily disturbed. If you are not careful, you may well make your sediment growth go to turbid simply by accidentally shaking it. However, there is a second type of bottom growth that you will encounter this semester. This type of growth is due to the presence of a slime layer around each cell that allows it to stick to other cells and the glass of the tube. Therefore, slime growth will lie on the bottom and be very hard to mix. Once you disperse it by mixing it, in fact, it may very well simply settle to the bottom in clumps.





1. What cultural characteristics are used to identify bacterial colonies?


2. Describe the appearance of a lobate colony.


3. Name and describe the three types of growth in nutrient broth discussed by your manual.





Obviously, a critical aspect of this exercise was getting successful single colonies in doing your streak culture. If you were unable to obtain good colonies you may have difficulty doing this analysis. In that case, do not just give up. Ask a colleague if you can share their results if they were able to get single colonies.


In activity 2 be sure to compare the two TSA plates with each other as well as the two NaCl plates with each other. In each case one species should grow "abnormally" compared to the other because of the difference the agar has on the species' growth pattern.




1. What was the fourth type of growth in broth we added?


2. What effect did the 1.5% NaCl have on the Proteus?


3. List the types of growth that each species gave you in the broth.




Unicellular Fungi: Yeasts





For the next few labs we will be examining microorganisms other than bacteria. In each case, there are two labs for each group of organisms. Therefore, we will discuss the entire group in the first module and review it in the second module.


The Fungi Kingdom was frequently placed in other kingdoms for many years due to its weird characteristics. They are basically eukaryotic, non-photosynthetic cells that get their nutrition through absorption through the cell wall/membrane. While some of them are distinctly unicellular (Module 15) others are composed of many cells (Module 16). However, these are not really organized enough to be called multicellular organisms like plants or animals.


In terms of basic non-reproductive structure, the kingdom can be divided into those that are unicellular (yeasts) and those that are composed of hyphae (molds, etc.). The hyphae are actually chins of cells that grow in length through true mitosis. Sometimes the cell walls will remain between the cells in the hyphae creating septate hyphae. Other times the cell walls will disappear shortly after the cell is formed creating a single long aseptate strand. If a number of these hyphae are wrapped together a mycelium is formed and this is easily seen with the naked eye.


When it comes to reproductive structures, the unicellular fungi may frequently use a process known as budding. This process is unlike mitosis as both the mother cell and the new daughter cell, usually only one at a time, continue to exist after the process is over. Here, a small bud simply starts to form off of the mother cell and grows to a certain size. When it reaches that size it simply breaks off, allowing the mother cell to continue to live and going off to grow to its full size and repeat the process. Filamentous fungi have many more variations in reproduction, both asexual and sexual forms. In the asexual category are any type of spore that is formed from a single cell. These will frequently be found on complicated structures that are described in your manual. Be sure to know these terms and associate them correctly with each type of fungi you examine. Sexual spores are those that are reproduced when two strains of the same fungi come into contact and, from their union, arises a sexually produced spore. Due to their simplicity we do not use the term male and female. We usually refer to them as (+) and (-) strains. These types of spores are actually the basis by which we classify the fungi as discussed in the manual. Be sure to understand these divisions or classes (depending on how your manual classifies them) and which type of spores each group produces.


This is the first exercise where several things will be done. First, you will be looking at numerous prepared slides of different types of fungi. It is important that you devise some method for learning what each species looks like and how to distinguish it from the others you are responsible for. You may find that drawing elaborate diagrams works, or that making comments alongside the photographs and diagrams in your manual is best. Whatever you do be sure to allow yourself additional time before the test to examine the slides again. Do not rely simply on the single opportunity provided in this lab period.


Secondly, this is the first exercise were you will need to start connecting the specific names of the species with specific diseases or special characteristics of the species. This is very important in the medical profession as you will need to understand what pathogen causes what disease as well as any special things about it that might help you identify it, control it, or whatever. Again, how you go about learning this is up to you but you must be able to do so.


While we will usually be examining these organisms as potential pathogens, we will also be seeing that many microorganisms are beneficial to humans. Sometimes this will be because of things they do inside of us, other times, like today, it will be because of something they provide for us. In this case, we will be examining the production of alcoholic beverages. However, the same group of fungi also are involved in creating leavened or rising breads.


As we look at the filamentous fungi there are lots of terms that are important and that you will need to know. Page 144 lists many of these and you need to know all of them except for the following: aerial, coenocytic, diphasic, fruiting heads, rhizoids, and sterigma.


A final aspect of the module on the filamentous fungi will be the attempt to grow them on slides so that they can be viewed under a regular microscope while alive. As you will see, these types of fungi can get quite large, quite rapidily and will fill a petri dish in a day or two. As such they can be viewed under a dissecting scope but that does not allow you to see the details that you will see on the prepared slides. Unfortunately, the prepared slides contain dead fungi and some of the details are not visible. Therefore, we will grow them on small chunks of agar on top of a slide and then simply move the whole microculture to a microscope. Unfortunately, this small piece of agar will dry out very quickly in the incubator, before any significant growth can occur. Therefore, we will create a moist chamber in which to keep the microculture slide. This is simply a sterile petri dish with a piece of filter paper in it that we will keep moistened with sterile water. The water will maintain a high humidity level in the petri dish and prevent the agar from drying out.





1. What kingdom do the yeast belong to?


2. What feature distinguishes the yeast from others in its group?


3. Give two species of yeast and the diseases they cause.


4. Define pasteurization.


5. Define bloom in regard to the yeasts.





See next module unit.




1. Describe the differences between the two types of yeasts as seen in their colonies.


2. Did your pasteurization process work? How did you know if it did or did not?


3. Which of the two yeasts did the most fermentation?




Filamentous Fungi: Molds





See previous module unit.




1. Why does this group of fungi also have its alternative name?


2. On what basis are the fungal divisions named?


3. Name a disease-causing fungus and a fungus that is used in making food.


4. What is a microculture?





As you examine the slides of the different types of fungi keep in mind that, except for the differences between the two yeasts that you examine, you will be required to identify the organisms on the basis of their physical characteristics. Be sure you can find distinguishing characteristics, besides color, that will allow you to separate them on an exam. Color is usually a poor indicator as it frequently is the result of artificially added stains that may vary from slide to slide depending on what was used.


In examining the grape juice tubes compare the differences between the two species of fungi used. Remember that one is a pathogenic species in humans while the other is the yeast used in commercial preparation of breads and alcoholic beverages. Obviously one should be a better grape fermenter than the other. In examining the effects of pasteurization on grapes, remember that the bloom found on grapes does not refer to the flowers but to the naturally occurring microorganisms that begin to break down the grapes. This grayish looking material may contain fungi capable of naturally fermenting the grapes. However, if you were successful in [pasteurizing your grapes, little fermentation should have occurred in this tube. Finally, the tube that had the bloom removed via pasteurization and was then inoculated with the commercial fermenter should have produced the most gas and alcohol.


Notice that as you look at the yeast colonies and the filamentous fungi colonies, there are significant differences. The former tend to look more like bacterial colonies while the latter are significantly different. You should also observe that the yeast grow much more slowly than the filamentous ones which will frequently fill the petri plate by the time you examine them. Regardless of which species you used in these two exercises, be sure to examine the results that other students got, using different species, as you are responsible for understanding what each looks like.


If your microculture was successful you should be able to see distinct hyphae and fruiting bodies under the low power of the compound scope. Hopefully, you should be able to see if your species has the cells walls in the hyphae or is aseptate. If your fungi is aseptate, see if you can see a process known as cytoplasmic streaming occurring where the cell cytoplasm moves along the hyphae.





1. What are the physical differences between the fungi you and your partner grew?


2. What kept the microculture from drying out?


3. Give a way to distinguish each of the fungal slides from each other.




Protozoans: Sarcodines, Flagellates, and Ciliates





Modules 17 and 18 deal with the second group of non-bacterial organisms we will be looking at. These protozoans are in the Protista Kingdom and are true microorganisms being unicellular. They are eukaryotic and are usually heterotrophic with a few autotrophic species.


While many of these are free-living organisms, many are of medical significance, causing a wide variety of diseases. They usually exist in two stages, cysts and trophozoites. The cysts are the non-motile resting stages while the trophozoites are the adult form and are usually motile. In fact, the type of motility they exhibit is the primary basis by which they are classified. These modules contain excellent tables that list the important free-living and parasitic representatives that we will examine and you will be responsible for. The work will be exclusively on the microscope so, again, you will need to develop important skills in identifying these organisms on the basis of characteristic features. Much of the test on these organisms will simply be microscopes set-up with these slides. You will need to identify them, name them completely and tell what disease they cause if they are pathogenic.


Module 17 deals with the three phyla that have both free-living and parasitic representatives. These are all classified on the basis of the type of locomotion they display. Be familiar with the phyla, their method of movement and their representatives. You are also responsible for the material in the tables in these modules except for the material in columns 5 and 7. In addition, remember the more correct and specific disease names that will be given in class as the table occasionally names only the symptoms and not the specific medical disease associated with each.


With these first three phyla we will be looking at both living slides you will make and prepared slides that we will provide. It is a good idea to start with the prepared slides so you know what you are looking at as the living specimen material frequently contains other types of protozoans that are there by accident or to serve as food for the specimen we are after. While you must look at both the living and the prepared slides, when they are available, remember that we will be using only the prepared slides in the exam, so concentrate on those.


In module 18 we will deal with a phylum that goes by several names: Sporazoa, Haemosporina and Apicomplexa. You may learn whichever you like but the last two are more current. This group is different in several regards. First, there are no free-living representatives, all are parasitic. Second, they are not motile. Third, they all have spore forms and, finally, all have complex lifecycles. The primary one we will study will be the Plasmodium, the causal organisms of malaria. Study the lifecycle that, in this manual, is divided into two sections: one showing the portion in the human, the other illustrating what happens in the vector, the mosquito.


In the prepared slides you will be looking at, you may see any of the forms shown to be in the human but none of the forms that are found only in the vector. Therefore, it is important that you look carefully at the diagrams of the sporozoite, merozoite, gametocyte and the several forms that are found inside the erythrocyte. Each of these is fairly distinctive.




1. What are the two stages in the lifecycle of a protozoan?


2. Define parasite.


3. How are the various protozoans separated into phylums?





See next module unit.




1. List the phyla of protozoans and give one parasitic representative for each.


2. List two vectors associated with protozoans and the diseases they transmit.


3. Give a distinguishing microscopic feature for two free-living and two parasitic protozoans.





Protozoans: Haemosporina





See previous module unit.




1. Why is this group not discussed with the groups in module 17?


2. What stage of the plasmodium is injected into the human?


3. What stages are actually inside the RBC?





As you review the material from these two modules, be sure that you understand the phyla, their characteristic traits, and the representative organisms we discussed. Much of this material will also be discussed in lecture so that will give you additional exposure to it.


The information here on the specific diseases is not to be as detailed as what you will need to know for lecture exams. Here we are primarily interested in your being able to provide the causal organism, classify it and tell the disease that it causes. Most importantly, however, you must be able to identify the pathogen under the microscope. This is difficult, but not impossible. More than likely it will require that you spend additional time in the lab looking at the organisms again, drawing accurate pictures to refresh you mind and then, perhaps, teaming up with a partner and setting up slides for each other to see how well you really remember them.





1. How can you microscopically distinguish between the Plasmodium and the Trympansoma?


2. Name 2 other disease-causing protozoans in this group.


3. What stages of the Plasmodium do you think you saw?





Some Platyhelminthic Infestations of Humans





In Modules 19 and 20 we will look at a group of organisms that definitely do not meet the definition of a true microorganism; in fact, these organisms are actually in the Animal Kingdom which means they are true multicellular organisms. However, since they are frequently pathogenic and usually very small, especially in their infective stage, they are usually discussed in an introductory microbiology course such as this.


The first group is the Phylum Platyhelminthe which are also knows as the flatworms. This is further broken down into classes, of which two are important to medical microbiology. The first of these is the Class Trematoda which consists of the flukes. These organisms are flat but tend to be shorter and broader than the next group. They frequently have oral and ventral suckers which allow them to feed and attach to their host. These typically have fairly complex lifecycles with several hosts and various larval forms that infect the different hosts. Be sure, when you are examining the slides, that you realize what stage you are looking at or it can get very confusing. For some of these you will need to know the adult and the egg or ova form as both can be useful in diagnosing the problem your patient is having.


The second class is the Class Cestoda which is made up of the tapeworms. The tapeworms are flat and very thin and long. Some of them may get up to sixty-five feet in length but, because of their thinness, they can easily be held in one hand. While you will be examining one tapeworm which will fit entirely on one slide, Echinococcus, the majority of tapeworms are too large to fit on one slide. Therefore, you will be looking at separate scolex and proglottid slides in order to see the components of a typical tapeworm. The scolex is the head and the proglottids are the individual body segments that make up the majority of the tapeworm. Again, you will also be examining the ova and larval stages for several of these organisms so be sure which it is that you are looking at. Do not examine any stage that the instructor does not require you to know.


The table in this module summarizes the organisms you will need to know and the diseases they cause. In the case of the Taenia spps. you will not need to be able to distinguish between the T. saginata and T. solium on the basis of the slides. In fact, what you will probably be examining are the parts of T. pisiformis which is commonly known as the dog tapeworm.


Related to these, in terms of also being animals, are the roundworms in the Phylum Nematoda. These will be discussed separately in the next help module.





1. Are the platyhelminthes true microorganisms?


2. What are the two classes in the phylum?


3. What are the two parts of a tapeworm?


4. What organs are usually infected by the hydatid worm?





Be sure that you are able to recognize the different pathogens discussed here in whatever stage you were required to look at. You will need to know the ova stage, larval stage and adult stage for several of these. you will also have to classify them and connect them to the disease they cause.


Rest assured, however, that the instructor will try to pick only those specimens that are truly representative of the species and the illustration in the manual.





1. What is the distinguishing microscopic feature of the Schistosoma mansoni ova?


2. Which of the organisms you looked at are too big to fit in one view under the scope?


3. Which did fit in one view?




Some Nematode Infestations of Humans





While the previous module dealt with the flatworms, this one will discuss the roundworms. These are also called nematodes and are placed in the Phylum Nemotoda or Nematyhelminth. While these at first look may look like common earthworms, they are different in several regards. The most significant of these is the fact that they do not have the segmented body that we associated with the earthworms. As you will see, there is also a tremendous amount of variation in length of this group of worms.


This variation of length is something you will need to be careful about. While it can be used as a useful technique for differentiating between some of the species, it can also prove problematic due to the variation that may occur within a species. Therefore, it is best if length is used only in certain cases and if other distinguishing features are identified. Be sure that when you examine Figure 20-1 you keep in mind Figure 20-3 and the measurements given in the first figure. This will help you understand the differences in length between the organisms you are responsible for.


Again, you will need to know both the adult, larval and ova forms of the organisms, depending on the species. However, it will not be necessary for you to be able to distinguish between the two sexes for any of these.


Be sure to study Table 20-1 and to be familiar with the organisms, their classification and the diseases they cause. The Discussion section of your lab manual will provide additional information of use to you as will your lecture component on these organisms.





1. What is the principle difference between the platyhelms and the nematodes?


2. Which nematodes represent the two extremes in terms of length?


3. How is a human usually infected with hookworm disease?


4. What form of Trichenella spiralis is found in the muscle tissue.?





Be sure that you find distinguishing features for each of the nematodes you need to be able to recognize. Sometimes it will be the overall size of the organisms, other times it may be the changes in diameter along its length, or the shape of the mouth parts. With the eggs, you need to be able to identify them out of the numerous other objects that show up in the typical fecal smear that they are found in.





1. What is a distinguishing feature of the Trichuris ova?


2. Which nematode did we examine only as a preserved specimen because it was to big?


3. Which species had the big diameter difference along its length ?





Preparing a Bacterial Smear





Since the organisms we will be concentrating on in this class, the bacteria, are so incredibly small, being able to properly use a microscope is, as you have seen, very important. however, in many of our labs you will also need to become proficient in making your own slides and in staining the organisms so that they will appear. Module 21 is the first step in creating a good slide and you will put this technique into use each time you make a bacterial slide in this class.


While we will use a slightly modified version of the method discussed here, it is important to realize the proper way to perform this technique. Be sure you understand the need for a clean slide and the best way to obtain one. This will usually not be a problem in this class as we usually dispose of our slides and start with brand new slides each time to reduce the possibility of contamination. However, another problem may arise because of the newness of the slides. Frequently these slides will stick together and you may find yourself unable to properly focus because the two slides together make it too thick. Be sure that you just remove one slide at a time from the box.


The modifications that we will make to this technique start with the use of a wax pencil to draw a straight line about 1/3 of the way along the slide. This line will help you know which side is up, will give you something to focus on initially and will help you locate the bacteria you have added. The second variation occurs when you go to air dry the smear. Rather than waiting 30 minutes for true air-drying, we will cheat a bit by holding it high above the Bunsen burner so that it will dry in a shorter time period.


The critical portion of this technique really occurs during the "fixing" portion when the bacteria are killed and made to adhere to the slide. Since we will frequently be using potentially pathogenic bacteria and since we will not be using any artificial "glues" or coverslips on the slides, this technique is very important. Top do this correctly is as much science as it is art so do not be frustrated if it does not work well the first time.


After the drop of water has air dried, move the slide through the hottest part of the flame three times, moving at a moderate rate of speed, as demonstrated by the instructor. If you go too quickly it will not be heated enough and the bacteria may still be alive and will probably wash off during the staining procedure. If you go too slowly, they will probably be heated too much and the cell walls and cell membranes will be destroyed resulting in an appearance like melted crayons and improper staining.


Be sure that you do one slide at a time, then stain and examine each. This is different from the instructions in the manual but will allow you to check your technique immediately and help you avoid having made the same mistake on all the slides you do initially.





1. Describe an ideal smear.


2. What are two common problems that might arise with a dirty slide.





One of the problems you most likely encountered in this module is making the smear either too thick or too thin. If you used an agar slant and transferred a loopful into a drop of water on the slide, you probably ended up with clumps or layers of bacteria that did not allow proper staining. It may even have been too thick to see through. However, if you went with a broth, you may have ended up having a hard time finding enough, or any, bacteria to examine. These are typical problems and do not reflect on your skills.


However, it will be necessary to overcome these problems in the future so that you do get good slides regardless of what you start with. If you are working from a slant it is important that you transfer only a small amount to the drop of water and that you spread it around as large as possible. If you are working from a broth, be sure to take your loopful from the most turbid portion of the broth, where ever that may be. Therefore, it is usually not a good idea to mix the broth before taking the sample.





1. Was your first smear a success?


2. How did you know if it was or wasn't?


3. What does it mean to fix a smear?


4. Which gave the best results -using a broth or a slant? Why?





Simple Stain





Now that the slide has been properly prepared, it is necessary to stain it so that it is more easily seen. Otherwise, the small size and the clear appearance will make it very difficult to see, let alone distinguish any parts of, the cell.


Stains that are typically used fall into one of two categories: acidic or basic dyes. Acidic dyes are those that have the colored ion on the negative portion of the molecule and, therefore, adhere to the positive components which are the basic portions of the cell. These are good for staining the cellular components inside the cell. However, what we are usually interested in seeing is the outside of the cell and its basic shape. Since these are negatively charged, we need a dye that is on the positive ion of the molecule. These dyes are in the Basic category of stains and are the most commonly used stains. The majority of the dyes that we will use in this class will fall into this category.


Each of the stains has a certain strength or affinity for the cell wall and can cause improper staining if left on for too long or too short. Therefore, be sure to leave the stain on for only the time noted and that you have the rinse water bottle handy when you add the stain as you do not want the time to expire and then have to look for the bottle to rinse it off.


Be sure to examine other students' slides as you will each be using different stains and bacteria species. Examining different slides will give you a better understanding of the appearance of the different stains and shapes of bacteria. This is a good time to refresh your memory regarding the three basic shapes of bacteria and their correct scientific terms.





1. Why are stains used?


2. What are the differences between a basic and an acidic stain?


3. Which sticks best to a bacterial cell wall? Why?





Basically, you will know that you have a successful slide if you see the bacterium that you added to the slide, if it is all stained one color and if there are at least some portions where the cells are far enough apart to distinguish their shape and growth patterns. If you do not achieve these goals then it is important to access what might have gone wrong so that you are able to improve your technique.


The most common problem, as stated in the previous module, will most likely be that the smear is either too thick or too thin. Again, if this is the case, reexamine the smear technique from Module 22 and rethink your technique. Then try another smear and stain to see if you can correct your technique.


If the smear is done correctly, the staining should go well as it is pretty straight forward. however, if the stain is too light or too dark it may be due either to improper timing or improper rinsing. If it is too light you may not have left the dye on long enough or you may have washed it too vigorously. Conversely, if it is too dark you may have done just the opposite. Again, rethink your results and try to avoid these mistakes with your next slide.


Be sure to examine other students' slides and compare them to yours. You should see differences in colors depending on the stain they used and in shape, depending on the species they used. You may very well find yourself using the stains and species they used today in the future so begin to learn what they look like.





1. List two types of basic stains we used and give their colors.


2. List 2 bacteria you saw and give the correct term for their shape.





Gram Stain





While the use of just one stain is called a simple stain, the use of multiple stains in the same techniques is usually refereed to as a differential stain as different cell types will stain differently. These are useful for distinguishing between different cell types which can frequently be useful in diagnosing a patient with a certain type of infection. In these techniques it thus becomes very important to get a smear of the correct consistency so that the entire smear stains one color or the other. This way you will know that any differences are due to the presence of two types of cells and not your poor technique.


This particular differential stain, the Gram Stain, was accidentally invented by C. Gram and it is still uncertain as to exactly why some cells stain one color and others stain the other color. The current theory is that it has to do with the differences between the cell wall in that Gram (+) cells have rigid protein walls while Gram (-) cells have a cell wall with a greater lipid concentration. This makes it difficult for stains to adhere to the Gram (-) cell walls so the dyes are easily removed.


The process begins with the preparation of a good smear as done in previous modules. Then a four reagent process is done that begins with an initial stain (crystal violet) which will stain both types of cells blue. A mordant (iodine) is added which will cause the initial stain to stick better to the gram (+) cells. However, when the decolorizer (acetone-alcohol) is added, it removes the dye from the gram (-) cells leaving them clear again. This means that when the counterstain (safranin) is added, the gram (-) cells are able to take it up and turn red. The gram (+) cells, however, are already stained blue so they are unable to take up the red stain and remain blue.


There are a number of things that can go wrong with this process and they are listed in the module discussion. Be sure to go through that list to avoid those problems and recheck it if you get a poor stain.


More importantly, from a clinical setting, there are also a number of important things that are usually different between the two types of cells besides the nature of their cells walls and the color they turn. The list in the module discussion is an important one because, after doing a simple, quick gram stain, you immediately know a variety of things that can help you grow the organism, kill the organism, know what problems it may cause or identify it.


Again, after you have successfully performed and examined your stain, be sure to look at your partner's slide so that you can see the opposite reaction.





1. How was this process discovered?


2. What is the most likely reason for why the two types of cells stain differently?


3. List 4 things you need to watch out for to insure a good gram stain.





Hopefully, you were able to get a successful stain the first time around. If you were unable to, be sure to go over each of the steps and the list of possible errors in the manual. The most common problem usually involves the thickness of the smear as this greatly affects the ability of the various reagents to get into all areas of the smear. You, therefore, end up with patches of red and patches of blue. When this occurs you are not sure which color is correct or if you happen to have a mixed culture of gram (+) and gram (-) cells. Finally, too much decolorizer or leaving too much water on the slide after each rinse can also cause problems for beginning stainers.


Since our emphasis in this class is on the medically significant aspects of microbiology be sure to learn the physiological and cytological traits of each type of organism. Remember that the majority of cells are going to stain red as most are gram (-). However, as listed in your manual, some very important pathogenic bacteria are gram (+). This technique, along with recognizing the shape of the bacteria, is also critical to simply identifying organisms found in your patient, as we will see in later modules.





1. Did you get a good stain on your first try?


2. If not what do you think went wrong?


3. Did you have any clumps of cells that were a different color than the rest? Why might that happen?


4. Why is this technique important in microbiology?


5. Besides whether something is positive or negative and what kind of cell wall it has, what do you know about a cell that stains pink?





Acid-Fast Stain





This staining technique is also known as the Ziehl-Neelson stain after the men who refined the process first developed by Paul Ehrlich. These microbiologists were interested in finding a quick and easy method to identify the presence of Mycobacterium tuberculosis, the causal agent of TB, in patients. These cells, as well as a few others, have a very thick lipid layer in their cell wall and this accounts for their color after the process. Most cells, including human cells, do not have this lipid layer and stain the opposite color they retain. Therefore, if a doctor simply takes a specimen of the patient's sputum and creates an acid-fast stain from it, the non-acid-fast human cells will stain blue while any M. tuberculosis cells present will stain an acid-fast red.


As with the gram stain, four steps are used to create the differences seen between acid-fast and non-acid-fast cells. There is an initial stain (carbolfuchsin) that will stain all the cells red. Next, a mordant is used that is not chemical in nature but physical. In this case, heat is used to drive the initial stain into the lipid cell wall where it is permanently bound. We will use a different procedure than explained in the manual as their method is difficult and potentially dangerous. Instead of holding the Bunsen burner over the slide we will place the slide over a beaker of boiling water. This will perform the same function but in a better way. Just be careful in handling the slide over the steam as the steam can scald you. Use tweezers or slide holders to manipulate the slide over the heat.


The decolorizing technique is also more severe than in the gram stain procedure. Here a stronger acid-alcohol reagent is used and the decolorizer is allowed to sit on the smear and then added again until the color stops coming off the slide. This harsh treatment removes the initial stain from the non-acid-fast cells but not from those cells that have absorbed the stain into their lipid layers. These decolorized cells are then made clear again and will accept the counterstain (methylene blue) when it is added at the end.


Thus, acid-fast cells end of red while non-acid-fast cells will be blue. Again, be sure to examine the slides of your neighbors so that you can see both types of cells.





1. Who discovered this technique?


2. What is it good for?


3. Why does it seem to work due to cell structure?


4. What is the other name for this technique?





Hopefully, by now you have gotten the smear technique down so that you are getting nice evenly stained slides without the patches of red and blue. If you are still having troubles with this aspect of the technique, talk to your instructor about possible remedies to your technique. You will be heavily dependent on a proper staining technique in your unknown procedure so you need to have this down pat.


If you did not get a correct stain with a good smear, examine the heating and decolorizing techniques as these are the most likely source of the problem. During heating the slide must be kept hot for at least five minutes and you must continually add the initial stain to prevent the slide from drying out. You also need to be sure that the decolorizing is done until any red color stops coming off but that no additional reagent is added beyond that time.


While we now have better techniques for determining if a patient is dealing with TB, this is still an important technique from a historical point of view and is still used in many countries. It is also a definitive test to supplement the other more modern diagnostic techniques in use today.





1. In terms of color result, how does this compare to the gram stain test?


2. Which species. was positive and which was negative?


3. List the steps in the process.





Cultivation of Anaerobes and Microaerophiles





The next series of exercises will deal with factors in the natural environment that affect the growth and survival of microorganisms, concentrating on bacteria. As we have seen in lecture, there are many things that affect the ability of an organism to grow and live. We will examine several of those in lab studies. In each case we must remember that if we want an organism to grow we would provide what they needed and if we wanted them to perish we would not provide them with what they needed.


This module examines the relationship between bacteria and their oxygen needs. There are basically four types of bacteria based on how they make use of oxygen. The obligate aerobes are those that have to have oxygen to survive. The obligate anaerobes are those that must have an oxygen-free environment as oxygen is lethal to them. In between these two are the facultative anaerobes which do not require oxygen but can tolerate its presence. Finally, there are the microaerophiles which need oxygen but in lower than normal concentrations and usually prefer high carbon dioxide concentrations, also.


Since each of these categories contain pathogenic bacteria, as listed in your manual, we need to have ways to provide the correct environment to culture them in when we need to identify them from a patient sample. Conversely, once we have determined their identity we may need to provide a harmful environment to them in order to get rid of them or prevent their growth in the future.


When it comes to growing obligate aerobes and facultative anaerobes, things are pretty simple as the standard petri plates, broth tubes and agar slants that we use are all designed to allow oxygen to reach the cultures. However, when we want to provide an oxygen-free environment we need to make use of several special techniques. One of these is to use a special chemical in the media that is capable of removing oxygen. Thioglycollate is such a chemical so, if you use thioglycollate broth, it will all start as anaerobic but, overtime as the oxygen is diffused into the surface layer, it will eventually develop three distinct layers as shown in your manual.


Another technique for removing oxygen from an environment is to use a Brewer anaerobic jar. This device is made up of a tightly sealed jar and some kind of a packet that contains material that will absorb oxygen and release some type of inert gas to take its place.


Finally, a microaerophilic environment can very easily be created by using an old glass jar and a candle. Remember that a candle consumes oxygen and releases carbon dioxide. Therefore, if we place the cultures into a jar, place a lit candle inside it, and seal it up, the candle will consume most of the oxygen but, before it is all gone, it will go out and leave a low oxygen, high carbon dioxide environment, perfect for growing microaerophiles.





1. What are the four groups of microbes based on their oxygen requirements?


2. How do you make a candle jar?


3. List two important pathogens that are obligate anaerobes.





As you examine the growth of each species in the thioglycollate broth, notice where the growth occurred in relation to the pink aerobic layer at the top of the broth. If the growth is in that area the bacterium is an obligate anaerobe. If it is at the interface of the pink area and the yellow area, it is probably a microaerophilic. The two types of anaerobes will be found throughout the broth if it is a facultative anaerobe and only near the bottom, below the oxygenated area, if it is an obligate anaerobe. Check with your instructor to see if each species grew where it should have.


The Brewer Anaerobic Jar is the best way to grow small amounts of facultative and obligate anaerobes. If they fall into this category, they should grow well here. However, if they are obligate aerobes, there should be little or no growth present.


The candle jar creates the best environment for the microaerophilics, of which Streptococcus pyogeness a perfect example. Therefore, it should grow better inside the candle jar than outside it. However, both plates should show some growth. This microorganism is the cause of strep throat so it is normally grown in a candle jar, or a similar device, after a culture is taken from a patient's throat.





1. Going back to exercise 13, relate the types of growth in broth with their oxygen requirements.


2. For each of the 4 bacteria used, give their oxygen requirements based on your results. Compare that to what they really are.


3. How does thioglycollate cause all the different types of environments in one tube?




Effects of Temperature





As has been explained in virtually every biology class you have had, temperature is a significant factor in the life of any organism. This is primarily due to the action it has on the proteins in the cells of the organism. Remember that proteins are important structural components, so too much heat will destroy those components, and that they also make up enzymes, which are necessary for all biochemical reactions occurring in the cell. Enzymes are designed to operate at certain temperature ranges and if they are placed in environments that are either too warm or too cold they will first operate less effectively and then operate not at all.


For this reason, bacteria can be grouped according to the temperature regimens they prefer. If they are adapted to colder temperatures they are called psychrophiles. If they grow best in the middle range of temperatures, such as the human body's temperature, they are called mesophiles. Finally, if they have special enzymes and proteins that allow them to function at higher than normal ranges, they are called thermophiles.


Understanding the temperature range for each bacterium will help you to provide the best environment if you wish to grow the organism or the worst type of environment if you wish to prevent its growth. Obviously, if you are dealing with a thermophile you will need to provide a much higher temperature to destroy it than if you were working with a psychrophile. Microbiologists try to determine the temperature limits under which a bacterium can operate in order to understand how to grow or control them.


There are several aspects that affect the action of heat on bacteria. Obviously, the species will be a factor as some are designed for different temperatures. Since some species produce resistant spores and others form clumps or clusters, this will all affect their susceptibility to heat. The amount of heat and the length of time they are exposed to heat are also important factors. Two terms are used in explaining the relationship between these two factors. Thermal death point (TDP) refers to the temperature an organism is killed by after a set time period, usually 10 minutes. This is the aspect we will examine today. Thermal death time (TDT) is used to refer to the time an organism dies after being exposed to a set temperature. Organisms will have different TDP's and TDT's depending on which temperature category they fall into.


We will be doing this experiment slightly differently from the manual's version in order to be more accurate and more efficient. Scattered around the lab will be five water baths set at specific temperatures. Instead of placing each tube into a beaker of water and chanting the water temperature, you will simply place each tube into the proper water bath before inoculating them onto the agar plates.


Besides survival and growth, temperature can also affect other aspects of a bacterium. We will examine the effect that temperature has on the production of pigments which are related to proteins either because they are composed of proteins or because they require certain enzymes for production.





1. List the 3 types of organisms according to temperature requirements and give their ranges.


2. Distinguish between TDT and TDP.


3. What is the relation between pigments, proteins and temperature?




The first activity is rather straightforward as you should see either a pigmented culture or a non-pigmented one. Whichever temperature produced the pigmented culture is the temperature required for production of the pigment. The other temperature will probably not cause a significant change in growth but is sufficiently different to change the ability to produce the pigment.


As you examine the plates with the streaks from the different temperature water baths, remember that each streak represents a group of bacteria that were placed in a different temperature for 10 mins. If they were not affected by that temperature regime then you should see growth. If, however, the temperature was too hot for them, you should not see any growth. Whichever is the highest temperature at which you observe growth will be the TDP for that species, within the parameters of this experiment. You should then be able to place each species into one of the three temperature categories dependent on what temperature they were able to grow at.





1. What temperature caused pigment production in Serratia marcescens?


2. On the basis of your tests, determine the temperature groups that Escherichia coli and Bacillus subtillis fit into.


3. How did we modify the temperature experiment?





Effects of Ultraviolet Radiation





Radiation may seem like an abnormal environmental factor but when you consider that, technically, all types of energy radiated from the sun is considered radiation it is a common factor indeed. The type of radiation we will concentrate on is the ultraviolet radiation that is in the ???nm range. These wavelengths are just the right size to enter the cell and disrupt the DNA in the cell. It does this by forming a dimer mutation that is characterized by the breaking of adenine to thymine bonds and the formation of thymine to thymine bonds. These types of mutations are destructive to the DNA and result in the death of the cell. incidentally, this is also the type of radiation that causes sunburns and tanning and it is the destruction of the skin cells' DNA that is causing this.


However, due to the nature of this type of radiation, several factors can affect how deadly this radiation is to the cell. Because radiation falls off rapidily as the distance between it and the organism increases, the cell has to fairly close to be affected by the radiation. Secondly, the longer you expose the cell to the radiation, the more effect it will have on it. Thirdly, this type of radiation is easily blocked by almost any type of material, including glass and plastic. Finally, some species are capable of repairing the damage to their DNA, to a certain extent, and so can survive the initial affects.


Because of the possibility of damage to all cells, it is very important that you do not expose yourself to the UV light while performing this experiment. This is particularly true of your eyes which are extremely sensitive to these wavelengths. Our set-up is designed to make this extremely difficult to do, however, if you really tried you could so don't!!





1. Ultraviolet light is a form of what?


2. How does it kill a cell?


3. What is the difference between bactericidal and bacteriostatic?


4. What factors affect the effectiveness of UV light?





As you examine the results of the experiment, notice where growth occurred and where it either did not occur or was much reduced. Then go back to what you did for each plate and consciously make the connection between cause and effect. You should see that plate four, which was not exposed to the UV light, probably had great growth. The other plates should have varying degrees of growth and, for plates one and two, there should be a difference between the half of the plate that was covered and the half that was exposed.





1. How did you know if UV light worked against the bacteria?


2. How did covering the plate with paper affect the UV light's effect?


3. Did the plastic lid have the same effect?



Effects of Disinfectants and Antiseptics





While the previous factors discussed have all been naturally occurring environmental factors, humans have developed a number of artificial factors to affect the growth and survival of bacteria. In this module we will examine two groups of chemicals that humans have made in the laboratory that are used against microorganisms. These chemicals fall into one of two categories depending on their harshness and strength. The first group are the antiseptics that are mild enough to use on living tissue while the second group are the disinfectants that are too strong to use on anything but inanimate objects. In either case, these chemicals could be either bacteriostatic in that they simply inhibit the growth of microorganism or bacterocidal if they actually kill the cells.


The first of these was carboxylic acid, also known as phenol, and it was used by Joseph Lister in the first attempt to control infections in the hospital setting. Since then, numerous other artificial chemicals have been discovered and your text book lists many of them. Your lab manual, however, lists only four groups with representatives that you need to be familiar with. Phenol is still used today both as a disinfectant and as a way of measuring the relative strength of other antimicrobial agents. The Phenol Coefficient is done by comparing the effectiveness of other agents to phenol, which is given the coefficient of 1. Anything with a number higher than 1 is stronger than phenol, while anything less than 1 is weaker than phenol.


These chemicals are tested in a fairly standard way that allows developers to test the effectiveness of new chemicals against bacteria and as compared to one another in a relatively simple and efficient manner. The technique is usually called the agar diffusion method and makes use of the ability of the chemical to diffuse out from a central area into the agar. As it does so it comes into contact with the test bacteria and either kills them, inhibits them, or does nothing to them. In either case a distinct appearance is seen surrounding the disk that originally contained the chemical and measurements can be made to compare the effect of one chemical to another. Obviously, the more successful antimicrobial agents are those that will leave the largest diameter kill zone around the disk.


However, if you remember anything about the process of diffusion from intro biology classes you may recall that there are several factors that affect the rate of diffusion. Several of these will not be of import to this experiment as each of the chemicals will be kept at the same temperature, which can speed up or slow down diffusion, and the experiment will be run for the same length of time, removing the effect of time on diffusion as a factor. However, the concentration of some of these chemicals will vary and this may affect the diffusion rate and some of these are larger molecules than others and this will slow down their diffusion rate. However, these factors are not sufficient to reduce the effectiveness of this test as an introductory test.


To make this test effective you need to be sure that you do a very complete job of streaking for confluent growth. This is a very different type of streaking than you have done before as the object here is create a dense, complete covering of the plate with the test organisms. Secondly, you need to be sure to use the same test chemicals against both organisms so that you can see any differences between the species and how they react to the different chemicals. Remember back to the module on gram staining and the differences between gram (+) and gram (-) cells in the way they are affected by antiseptics and disinfectants.





1. What was the first antiseptic and who started to use it?


2. What is the difference between an antiseptic and a disinfectant?


3. List 3 types or categories of disinfectants and a specific one in each group.





Again, it is important that you examine the two plates together and compare the effect that each chemical had against each species of bacteria. If you were able to get a complete coverage of the plate with the bacteria there should be distinct zones of inhibition around at least some of the antimicrobial agents. By measuring the diameter of the zones you can determine which agent was most affective and how effective it was against each species. At this point you need to determine the gram staining group of each species used and see if your results confirm what was reported in the gram staining module.





1. How were you able to tell if a chemical was affective?


2. For each bacteria used, list the most effective chemical against it.


3. Define phenol coefficient.





Effects of Antibiotics





While the previous module dealt with chemicals that were originally created artificially by humans for use against many types of microorganisms, this module deals with chemicals that originally were created naturally by fungi and bacteria and are effective primarily against bacteria. Humans have then modified these and learned to make them artificially to produce the numerous types of antibiotics we now have available to us. Examples of fungi that make antibiotics include the Penicillium genus while Bacillus and Streptomycetes are examples of bacteria that make antibiotics.


The test used to test the effectiveness of antibiotics is a modification of the technique employed in the previous module. When it is used to test antibiotics, however, it is usually called the Kirby-Bauer Method. This test makes use of disks that are preimpregnated with the antibiotic, at carefully controlled concentrations, and a special agar called Mueller-Hinton Agar. Other than that, the technique is virtually identical.


However, when the petri plates are read following incubation, special care must be taken in examining the zones of inhibition that form around the disks. Table 32-1 in your manual lists the more common antibiotics along with a series of zone diameters. Notice that in most cases even a fairly significant clear zone around the disk may not indicate effectiveness of the agent against the bacteria. These variations are due to such factors as the species used, the concentration of the agent, the amount of inoculum, etc.


Another aspect of this test is the fact that many species of bacteria are developing resistance to many types of antibiotics. This is occurring for a number of reasons including overuse of antibiotics, improper use by patients, and mutations occurring in the bacteria. This is becoming an area of increasing concern for both microbiologists and medical professionals and will impact your career in the future.





1. What are the differences between antibiotics and antiseptics/disinfectants?


2. What is the alternative name for this technique?


3. Define confluent growth.


4. Name some bacteria that are showing drug resistance.





Again, as with the antiseptic/disinfectant test, be sure to examine the two plates next to one another and compare the results. Some of the antibiotics may be effective against one species and ineffective against another. Several antibiotics may be effective against one species but not equally so. Be sure to compare your measurements to the table in the module and record whether the organism was resistant, intermediately susceptible or susceptible to the antibiotics.


If you see any zones that are fairly clear but still have a few bacteria in the zone, you may be seeing examples of resistance against that antibiotic.





1. How were you able to tell if an antibiotic was effective?


2. For each bacteria used list the most effective antibiotic.


3. What are some differences between this technique and the one used for testing antiseptics?





Exoenzymes and the Major Food Groups





The next series of tests, from module 33 to module 39, are frequently used as tests in identifying an unknown microorganism. You will see many of these again as you use the BacterioIdent CD-ROM program to work on the unknown bacterium. They are used, in various forms, by clinical labs to diagnose the pathogens that patients are dealing with when a doctor sends in a specimen. They are therefore, very important to all aspects of microbiology.


The first module deals with three of the basic food sources for any organism: starch and proteins. AS with most of these exercises, the tests are dealing with the organisms ability to produce the enzymes necessary to metabolize the various organic compounds. These are frequently called exoenzymes since they are released outside of the cell. This allows the bacterium to break down compounds that are too large to be brought across the cell membrane and create smaller compounds that can make it through the membrane. Since it is the basic genes of the organism that give it the capability to produce these enzymes, these are terrific tools for helping to identify the bacteria.


In the first activity we will look at whether or not organisms can produce amylase which is the enzyme needed to digest starch. If they do produce this enzyme the starch will be broken down into maltose and eventually glucose. This is a type of chemical reaction called a hydrolysis reaction because, along with the enzyme, water must be added to lyze or breakdown the starch. We will be able to determine if this happens by adding iodine to the starch agar plate after incubation. Iodine reacts with starch to create a black/purple complex that will color the starch plate. However, if the bacterium has digested the starch immediately around its colony, there will not be any for the iodine to react with and a clear zone will be seen around the colony. This would indicate a positive starch hydrolysis test.


Activity two and three involve the metabolism of two kinds of proteins. In the first of these, the protein casein will be present in the Milk Agar. This protein is what gives milk its characteristic white color. If your organism produces caseinase, the enzyme required to break milk down, there should be a clearer area around the colony as the casein is hydrolyzed.


Finally, in activity three we will look for the ability to hydrolyze the protein gelatin by producing the enzyme gelatinase. This protein is what makes things like gelatin a solid at room temperature after it has cooled from being a liquid. If it is hydrolyzed the gelatin substance will not stay firm and you will have a liquid again. Therefore, a positive gelatin hydrolysis test will show as an agar that will not harden even after being cooled in ice. The technique used in this test is different from any other you have done as this requires a stab inoculation. The technique here is to take an inoculating needle, not a loop, and coat the bottom portion in the organism. The needle is then stabbed straight down into a gelatin deep until either the handle of the loop come close to the top of the deep or the needle itself comes close to the bottom of the test tube. It is important to check this test at 48 hrs. and at 7 days to see if your organism is a fast liquifier, a slow liquifier or a negative test. After pulling the tube out of the incubator, it may very well be liquefied simply due to the temperature in the incubator. You then need to place it in a beaker of ice, not just in the refrigerator as the manual says, for about 10 minutes. If a significant portion of the agar remains liquid you have a positive test. However if it all hardens you have a negative test. After the 48 hr. test, put it back into the incubator and repeat the test after 7 days.


We are not doing the fat hydrolysis test in this class.





For this module you will also need to read the individual activity intros to answer these questions-


1. Define exoenzyme.


2. What are the three major groups of chemicals used as food by bacteria?


3. What is involved in a hydrolysis reaction?





In examining the results of these activities the most important thing is remembering what a positive test looks like and what you have to do to test for it. The supplemental lab books available for you in the lab will help as they have photos showing positive and negative test results. With the starch plate, you first have to add iodine which should turn all areas containing starch a blue/black color. If no starch hydrolysis was done by the bacterium, everything should be that color up to and around the colony. If, however, your organism produced amylase, there should be an area that is not that color right adjacent to the colony. It will look like a halo around the entire colony.


The casein hydrolysis test is even easier to see as you do not have to add anything. Simply hold the plate against the light or a dark background and see if there is any change in appearance immediately around the colony. Sometimes this will be a very clear and spectacular clearing. Other times it may be just a thinning of the white in the plate as the caseinase has not had time to completely remove the white casein.


Finally, the gelatinase production requires immersion in an ice bath, being sure that it goes all the way to the top of the agar deep. If your organism produced the gelatinase, the gelatin should have been destroyed and the agar will not be able to harden. However, if the entire deep hardens after being in the ice it is a negative test. What frequently happens is that the organism is a strict or obligate aerobe and cannot grow well below the very top of the deep. This will result in a turbid top portion and a clear bottom portion. The top agar will have been degraded and will not solidify. However, the bottom has not been affected so it will solidify leading to confusing results. This is usually the result of a positive test that has not had time to affect the entire tube.





1. What reagent had to be added to complete the starch test?


2. How did you know if you had a positive or a negative casein hydrolysis reaction?


3. Which of these tests did we not do?


4. What does it really, exactly mean when your organism gives you a positive protein hydrolysis reaction?


5. Regarding oxygen requirements, what type of organism is most likely to cause just the top portion of the nutrient agar deep to test positive?




Carbohydrate Fermentation





While starch is in the carbohydrate group of compounds it is a complex carbohydrate or polysaccharide. Today's module deals with a bacterium's ability to produce the enzyme needed to break down simple carbohydrates or mono- or disaccharides.


The test involves inoculating various sugar-containing phenol red broths. The sugar is the substrate that the bacterium will attack and, in doing so, it may be able to ferment it producing either acids alone or acid and gas. If it does produce acid it will lower the pH causing the phenol red, a pH indicator, to go from red (neutral) to yellow (acid). If the process also produces gas, such as CO2, then bubbles will be trapped in the inverted Durham tube that is inside the broth.


While numerous sugars can be tested in this manner, we will just look at three:


Glucose, which is broken down by glucase

Sucrose, which is broken down by sucrase

Lactose, which is broken down by lactase





1. What is the general equation for a chemical reaction as given in the module?


2. What are the components of the fermentation tubes and of what use is each component?


3. What carbohydrates will we use in this exercise?





IF your organism gives you strong positive results, this is a fairly easy test to analyze. If your organism produced the correct enzymes, there should be a distinct yellow color to the broth and, if gas is produced in the process, there should be a visible quantity of gas trapped in the Durham tube. Remember, anyone bacterium may be able to metabolize one, two, three or none of the sugars tested. Also, just because it produces acid and gas with one sugar does not mean it cannot just produce gas with another and do nothing with a third.


There is also a good possibility that, for any of a number of reasons, you may get confusing results such as an orange color or a red outer broth and a yellow broth inside the Durham tube. These may be the result of either contamination, changes in the media itself, or inoculation with only a small bit of inoculum. In any case, this is frequently a sign that the test needs to be repeated.





1. Did all the organisms react the same way with all the sugars?


2. Pick one of the organisms used and explain how it handled each sugar.


3. What is a comparative control and what is it used for?




Nitrate Reduction





Because of its importance in the chemical structure of amino acids, proteins and nucleic acids, nitrogen is a critical element in the life of any organism, including bacteria. Many organisms are able to get it from the reduction of nitrate which is one of the most readily available forms in the environment. Some organisms cannot reduce nitrate at all, others can reduce it only to nitrate and some are capable of reducing it all the way to N2 or NH2.


While the test itself is easy to do, the completion of it is not due to the process required to analyze the results. After inoculation of the Nitrate media, two reagents are added: sulfanillic acid and alpha-naphthylamine (Nitrate A and B, in some cases). If your organism was only able to reduce the nitrate to nitrite, these reagents will react with the nitrite and produce a red color. A red color at this point ends the experiment.


However, if you organism does not elicit a red color at this point, powdered zinc is added to the media. Zinc reacts with the original nitrate and chemically reduces it to nitrate which then reacts with the previously added ingredients to form a red color. Therefore, if you get a red color at this point it means you have a negative nitrate reduction test since the nitrate was still present to react with the zinc. However, if you do not get a red color at this point either, it is because your organism was capable of reducing the nitrate all the way down to N2 or NH2. Thus, there is no nitrate to react with the zinc or nitrite to react with the sulfanilic acid or alpha-naphthylamine.


Notice that this complete process then requires two enzymes, nitrate reductase and nitrite reductase, for the complete reduction to occur.





1. What are the three possible results of this test?


2. What reagents were used in this test?


3. What occurs when a chemical is reduced?





This test is easily done and understood if the flow chart in figure 35-1 is understood. It is critical to understand that the two reagents react with only the nitrite that will be present either because your organism produced it from the nitrate or because the zinc produced it from the nitrate. Only the absence of any kind of red color indicates complete reduction.


While it does not occur too frequently, there is the possibility that, due to incomplete reduction of the nitrate and/or nitrite, the color development may be weak. This usually indicates that your organism is doing the reduction but has not had time to complete it.





1. What is a positive test result for nitrate being reduced to nitrite?


2. What exactly happens when zinc is added to the media? Why would that happen even if no organism had been added?




Urea Hydrolysis





As we will see in several experiments this semester, there are a large number of gram-negative bacilli that inhabit the intestinal area. Most of these are non-pathogenic, normal flora. However, several are deadly pathogens and we need ways to distinguish one group from the other. There are several tests, when performed as a group, that allow us to do this. They are thus useful for allowing a physician to determine if the problems the patient is having are due to the pathogenic species or some other problem. They are also useful in identifying which species of gram-negative rods we are dealing with as none of these species reacts exactly the same to all of theses tests.


Most of the non-pathogenic species are lactose fermenters and will test positive in any of the lactose fermenting tests that are available are used. However, the genus Proteus is an exception as it is a non-pathogenic non-lactose fermenter. Therefore, if you culture such a bacterium from a patient you may incorrectly diagnose Salmonella or Shigella when all you have is normal Proteus. However, this simple test will separate out Proteus from the pathogenic species.


This is because Proteus is the only one capable of hydrolyzing urea, a common nitrogen-containing waste product of most cells, into ammonia and carbon dioxide because it produces urease. Since ammonia is an alkaline compound, its production will create an alkaline pH environment which can be seen by the use of any one of several pH indicators. In this case we will again use phenol red which, as we have already seen, is red when neutral and yellow when acid. When it is in an alkaline environment, it turns a deeper red or cerise color. Therefore, such a color will indicate a positive urea hydrolysis test.


Another interesting factor in this test is that the urea media has to be handled differently from any other we have dealt with this semester as it is destroyed by high heat, as might be found in an autoclave. Therefore, we cannot sterilize it as we do the other media. If you remember back to our first modules, there are alternative ways to sterilize media besides heat. In this case, we usually use filtration sterilization to trap any contaminants on very fine filter paper before collecting the filtrate in a sterile flask below the filter. This sterile media is then poured into sterile test tubes for use in this test.





1. What group of bacteria is this test good for separating out?


2. What are some pathogenic bacteria in this group and the diseases they cause?


3. What is the enzyme that allows this reaction to occur?





This test is one of the more difficult to perform, perhaps due to the difficulty in sterilizing it. Frequently, contamination results from the filtration process, since it is more difficult to do than simple autoclaving. Many organisms also do not give strong negative or positive tests because of other by-products they may produce. However, when Proteus is used you will get a nice deep red color developing and a positive test is usually very evident.





1. How did you know if you had a positive test result?


2. What is the pH indicator used in this test?


3. How is this media sterilized? Why does it have to be done this way?




Litmus Milk Reaction





The Litmus Milk Reactions test is a very easy one to perform and a difficult one to analyze. It requires that you simply inoculate a tube of Litmus Milk Medium and check it at 24 hrs., 48 hrs., and at 7 days. However, because of the complexity of the medium and all the various things that it is designed to tell you, it can be difficult to interpret.


The main ingredients of interest are the milk, which contains both lactose and casein, and the litmus, which is a pH indicator and a reduction indicator. Some organisms will ferment the lactose producing acid and/or gas. The acid will turn the pH indicator a pinkish color and will attack the casein creating an acid curd. Occasionally this occurs so quickly that the curd, which is hard to begin with, squeezes out the excess liquid to form a clear, brownish fluid called whey on top of the curd. If gas is also produced this curd may appear broken and split by the gas pockets that form.


Other organisms will not ferment the lactose but, instead, will create a renin-like enzyme that directly attacks the casein causing a softer curd that occurs primarily at alkaline pH's. This turns the litmus a deeper purple/blue. Some organisms will continue to produce the enzyme until all the protein is broken down causing the entire broth to turn brown and opaque. This is called peptonization.


Finally, some organisms may cause reduction occur which will turn the litmus a white color. This needs to be approached cautiously as there will frequently be a whitish precipitation that accumulates on the bottom of the tube and this is not evidence of reduction.





1. What is the pH indicator used here?


2. what exactly occurs when an acid curd is formed?


3. Define peptonization.


4. What is the sugar in milk that is fermented in this experiment?





Be sure that you understand what the pH of the media was at the 24 and 48 hr. marks as this will assist in understanding what type of curd you have. Frequently, the pH will change during the remainder of the incubation and the red color, in particular, may disappear or become more difficult to see as time progresses.


In testing for the curd formation, tilting the tube gently from side to side, being sure that nothing comes out of the top, will help. Frequently, the results will be mixed. For example, part of the tube may turn a certain color, or begin to clear. This is where showing the results to the instructor will help in interpreting the results.





1. What are the colors associated with an acid and a basic pH?


2. List one species you used and explain the entire effect it had on the medium.


3. How do you know if your organism produced gas?





Hydrogen Sulfide Production





Many amino acids, and thus proteins, contain sulfur in them. Some organisms have the ability to metabolize these amino acids and release the sulfur, usually in the form of H2S gas. This gas is the rotten egg smell associated with swamps, chemistry labs, etc. Since we try to avoid directly sniffing gases in the lab and since the quantity of gas produced may be very small, we have an alternative way to detect the formation of H2S.


In this experiment, the sulfur containing amino acid peptone will be used. To detect the production of the gas, iron is also present in this media. This iron will react with the H2S gas to form iron sulfide, a black precipitate. Therefore, a positive test is seen in the presence of a black precipitate anywhere in the tube, usually around the colony.





1. What type of compounds contain H2S?


2. What actually causes the black precipitate to form in the media?


3. What is the complete name of the media used in this module?





Most microbes do not produce the enzymes necessary to digest these types of amino acids so most of the species should give you negative results. However, when a positive result occurs it is usually fairly easy to see. Problems may occur, however, if your organism is an obligate aerobe or if inoculation is not done correctly. In these cases, not enough growth may occur within the media and the precipitate may not occur or be visible.





1. Which bacterium created H2S?


2. Besides the black precipitate, how might you have been able to detect a positive test?


3. Why are aseptic procedures especially important in this module?





The IMViC Tests





This module actually deals with a series of four tests that are combined in the acronym "IMViC" which are used to separate out the gram negative bacilli that we have mentioned reside in the intestinal tract. As stated previously, some of these are normal, non-pathogenic flora while others are potentially serious pathogens. Since you cannot distinguish between them visually, tests are required to separate out the various organisms as they each react differently to the overall series of tests used.


"I" stands for Indole Production and refers to the ability of some organisms to take the amino acid tryptophan and break it down for energy. In doing so they produce indole, pyruvic acid and ammonia. To test for a positive indole production test we simply add Kovac's Reagent to the incubated tube and look for the presence of a red layer at the top of the tryptone broth. This is the result of a chemical reaction between the indole and the Kovac's Reagent. If no indole has been produced, no red layer will be seen.


"M" and "V" refer to two related tests called the Methyl Red- Voges Proskauer Tests. These tests start off by inoculating one MR-VP broth and then, after incubation, dividing it in two to do each of the two tests. The test is designed to see how you your organism handles the metabolism of glucose (dextrose). Some organisms cannot hydrolyze it at all and will give a negative reaction for both tests. Others will breakdown the glucose into an acid pH by-product which will react with the Methyl Red reagent to produce a red color. Others will continue to work on the acid byproduct to form a more alkaline product such as acetyl methyl carbinal. This will not react with the Methyl Red reagent but will react with the Voges-Proskauer Reagents (VP A and VP B or Barritt's Solution A and B or alpha-naphthol and KOH-creatine) to form a red color also. Be sure that you follow the instructions on separating the MR-VP Broth before adding the reagents because each one has to be added to its own test tube to avoid affecting the results.


"i" is there just to be able to pronounce it.


"C" stands for Citrate Utilization and refers to the fact that some organisms are able to use the sodium citrate in the media as an energy source. Since this is the only possible energy source in the media, one way to check for a positive test is to look for significant growth on the slant. However, a more accurate and reliable method is to rely on the pH indicator provided in the media. Bromthymol blue is yellow at the acid end, green when neutral and a cool blue when in an alkaline environment. If the citrate is degraded, it will cause the production of alkaline endproducts which will cause the media to turn blue.


Recording the results to all these tests, along with the results of the Urea Hydrolysis test, and the ability to ferment lactose will allow you to easily identify which gram-negative rod you are dealing with.





1. What does the acronym IMViC stand for?


2. What group of organisms are these tests designed to separate?


3. What reagents go with each test?





Care must be taken in examining the Indole Test as students frequently assume that any kind of layer after the addition of the Kovac's reaction is evidence of a positive test. Anytime Kovac's reagent is added to any liquid it will forma layer of some sort. The only one that indicates a positive test result is a bright red layer. Again, remember that the formation of such a layer indicates that that organism was able to breakdown the tryptophan and produce, among other things, indole.


With the MR-VP tests, most organisms will either give you two negatives or one negative and one positive since it is unusual for the organism to produce both an acid and a basic product from glucose at the same time. In many cases the color formed will not be a real bright red, more of a subdued pink may occur. This will sometimes lead to confusing interpretations so be sure and compare the two colors formed to each other, two any controls used and to the pictures provided in the supplemental books.


The only sign that should be taken as a positive one in the Cirtrate Utilization test is the one that shows up as a nice blue color change in the media. The amount of growth that occurs can be very subjective depending upon a variety of factors, including how much inoculum you originally added. Again, comparison to any controls available and to the photos in the books will help interpret whether or not your organism was able to survive by using the citrate as a food.





1. Why will the MR and the VP tests rarely both be positive for the same organism?


2. What pH indicator is used in the citrate test and what are its color changes at each pH?


3. What is a positive indole production test and what does it tell you about the organism?





Assay of Bacteria in Milk





Bacteria and other microorganisms are useful in the preparation of many types of foods that we enjoy everyday, especially when it comes to dairy products. However, they can also be the source of significant pathogenic organisms that can cause serious illness. In some cases these bacteria come from the cows themselves. In other cases they are introduced during the collection process as the milk producers gather and ship the milk. Finally, if the people processing the milk into the various endproducts are not careful they may introduce pathogenic bacteria. The manual contains references to a number of diseases that may be transmitted this way so be sure to examine that section.


Because of the possibility of these problems, the USDA and other government agencies are responsible for ensuring that the milk supply is safe. Standards have been created to inform the public about the status of the milk they buy. Raw milk is allowed to have up to 50,000 bacteria per ml. While pasteurized milk is restricted to under 30,000 bacteria per ml. However, these organisms are non-pathogenic and natural. What is of real concern is the number of coliform bacteria which represent fecal contamination. These must be below 10 per ml. Most milk sold in the US, however, contains significantly fewer numbers than any of these standards.


The reason there are so many bacteria in milk is that the process of pasteurization, originally developed by Pasteur for the wine industry, is not a sterilization process. Rather it is simply a technique to reduce the number of microbes so that the pathogenic ones are almost completely removed and the other types are simply reduced to the point where the product does not spoil too quickly.


However, with this significant number possible in a ml. Of milk, special techniques must be used to determine the number of bacteria per ml. Usually the pour plate technique is performed in conjunction with a serial dilution. This technique allows the original ml. of milk to be diluted by a known quantity so that when a number of colonies is found present in a pour plate, calculations can be accurately done to work back to how many microbes that would represent in the original ml. of sample. The technique for doing this is shown in figure 42-1 and should be followed closely.





1. How many organisms per ml. of milk could you be consuming if you drank a full 8 oz. glass of milk?


2. What is a serial dilution?


3. Why are sterile water blanks used in this process?


4. List the three sources of contamination in milk products.




The most important criteria for success in this experiment is to get at least several plates that have a "countable" number of colonies. If you have any question about what this constitutes or how to do this, review module 11 on the Quebec Colony Counter. Once you have plates that you can reasonable count, you will have to compute backwards to the original ml. of sample using the dilutions to multiply your number of colonies. Remember that each colony represents one original bacterium and that each succeeding dilution should end up with fewer and fewer bacteria, if done correctly. Figures 42-3 and 42-4 give you examples of how to compute the final results. As seen here, it is simply a matter of multiplying by a factor of 10 for each dilution done. Thus a 10-4 dilution means that you have diluted it by a factor of 4 so you would multiply the number of colonies in that plate by 10,000. If you were counting a 10-2 plate you would multiply the number of colonies found by 100 to get back to the original per ml. basis.


In your experiment you should see two significant differences. First, in either series there should be a decrease in the number of colonies per plate as you go through the dilutions. Secondly, if your pasteurization technique worked, the pasteurized milk plates should have fewer colonies, per similar dilution, than the non-pasteurized plate.






1. Diagram how you would perform a dilution, suing 9 ml. blanks, that would include 10-2 and 10-4 dilutions.


2. Did you get several plates in each series that had good, separate colonies on them?


3. Did pasteurization prove effective in reducing the bacterial count?


4. If you had a 10-6 plate that had 12 colonies on it, how many organisms were in the original ml. of milk?




Assays of Harmful Bacteria in Foods





AS we have seen in lecture, numerous pathogenic microorganisms are transmitted via food. Many of these are caused by bacteria that are introduced either from the soil the crops are grown in or from fecal contamination. This fecal contamination can occur indirectly through feces containing soil or directly through faces from the animals being prepared for food. Again, as with milk products, the end product may be contaminated because the food itself is contaminated, because it is harvested improperly, or it is processed improperly.


The discussion to this module mentions a few of the more common pathogens introduced into food in these ways. We will be examining two types of food for the presence of harmful bacteria. The first will be an animal, chicken, that might have been contaminated simply because poultry are notoriously dirty animals and the processing process can be very messy. The livers that we are using were bought as frozen livers. However, remember that these are usually found inside the body cavity of whole chickens, ducks and turkeys. Therefore, if we find that the livers contain contamination it means that the entire bird they are packaged in can be contaminated.


The second food group we will examine is the vegetables. Spinach grows very similar to lettuce in that it is low lying and is gathered with a great deal of dirt on it. If that dirt contains fecal matter, it is easily transferred to the vegetable and to the end product.


In either case, one of the first steps to take to insure that such contaminant does not ruin your day is to thoroughly wash the food prior to cooking. This will remove a significant amount of the contamination. Completely and quickly cooking the food and keeping it at the proper temperature, and immediately refrigerating any remaining, will also help prevent transmission.


The technique used here is almost identical to that employed in the previous module on dairy products. A serial dilution will be done, pour plates will be made and counts will be made to determine the number of bacteria in the original sample of food. Two differences will be seen, however. First, since we are not dealing with liquids here we will need to create a water/food blend that can easily be measured and transferred. This will require diluting a weighed out sample of the food with a measured amount of water to create the original dilution. Therefore, the blending process is the first dilution step. The second difference is that we will also be plating the results on EMB agar, along with the standard plate count agar. EMB is a selective and differential agar that is used to select for gram negative organisms and to differentiate between lactose fermenters and non-lactose fermenters.





1. List two examples of pathogens that can be transmitted via food.


2. What group of organisms are identified with the aid of EMB agar?


3. Why did blending the 20 grams of food with the 180 milliliters of water result in a 10-2 dilution? How can we relate gms. with ml. here?


4. Why does measuring out 1 ml. of liquid result in a 10-1 dilution while transferring .1 ml. of liquid results in a 10-2 dilution?





Again, the first step in a successful experiment here is to get several plates that have good, countable, separate colonies growing on them. Following the instructions on this module and the last module will help you interpret the results. Again, the main idea is to see if the food was contaminated by bacteria, which food was contaminated the most and if there were specifically some coliform bacteria present. This latter group will be identified by growth on the EMB agar. If they are clear colonies they have the potential to be non-lactose fermenting pathogens while if they are colored colonies they are most likely to be non-pathogenic bacteria representing normal flora. However, they still are used to indicate fecal contamination and are, therefore, undesirable.





1. Which food was the most contaminated? How did it have per ml.?


2. Did any of the plates have so many bacteria that they could not be counted?


3. What did the growth on the EMB agar look like?


4. Was the EMB growth evidence of pathogenic or non-pathogenic bacteria?





Ames Test for Chemical Mutagens/Carcinogens





Scientists now recognize a critical correlation between the ability of a chemical to cause a mutation and its ability to cause cancer. The ability to cause a mutation, a sudden inheritable change in the cell's DNA, is called mutagenesis and is caused by mutagens. These mutations apparently are involved in causing normal body cells to being growing abnormally leading to certain types of cancers. A cancer-causing compound is a carcinogen and is said to be carcinogenic. Obviously, there needs to be a way to determine if a chemical we are exposed to is mutagenic and carcinogenic.


In the past, such tests usually involved elaborate animal studies that took many years to complete and were very expensive. Recently, however, a special test using bacteria has been developed that allows researchers to recognize the mutagenic potential of a chemical in a matter of hours rather than years. Called the Ames Test, it involves the use of special media and special bacteria strains to test compounds for their ability to cause mutagens. If a compound is found to be mutagenic, it can then be shelved or investigated further.


The special bacterial strain used is called an auxotroph which means it cannot grow unless some special compound is present in the media. The one we use requires histadine in order to grow as it cannot make it on its own. Therefore, if the bacterium is grown in minimal agar, the special agar used here, that is lacking histadine, no bacterial growth should occur. However, this strain is also prone to back mutations which, when they occur, allow the organism to begin making its own histadine. Therefore, this mutated strain is capable of growing without histadine in the agar.


Therefore, if we grow the auxotroph in agar without histadine, expose it to the suspected mutagenic chemical through a simple agar diffusion technique, and it begins to grow, we know that a mutation has occurred and the chemical is indeed a mutagen.





1. What is a mutagen?


2. What does carcinogen mean?


3. What does auxotroph refer to?


4. Who invented this technique?





The main thing to check for here is the presence of growth around any of the disks placed on the agar. If there is growth it means that that chemical caused a mutation in the bacterial strain so that it can now grow without histadine. This indicates a mutagenic agent and a possible carcinogenic agent. If, however, no growth occurs around any of the disks it means that the chemical did not cause a mutation and is, therefor, probably not a mutagenic or carcinogenic agent.


Remember, however, that some mutations may occur spontaneously, just due to the complicated process of DNA replication. Therefore, the control reagent is used to show the number of spontaneous mutations that occurred which can then be compared to the number of induced mutations around the chemical agents. If there are twice as many mutations around the chemicals tested as around the control we can say that the chemical truly induced mutations greater than would have been expected with spontaneous mutations only.





1. Which chemicals gave evidence of being mutagenic?


2. Did the area around the control show much growth? What is that indicative of?


3. What is the relation between carcinogenic and mutagenic?





Bacterial Agglutination Tests





The next two modules, 46 and 47, will be used primarily for the information they contain and not for the actual experiments presented. Instead, we will do other experiments that convey the same material. The theme of both of these is antibody-antigen reactions. Antigens are the chemicals that all cells produce that are recognized by the human body as being foreign and against which the body produces antibodies. Antibodies are chemicals produced by our immune system that react with antigens in an attempt to destroy them in one way or another. The study of these reactions is called serology.


While there are several types of antibody-antigen reactions we will examine only two in these exercises: agglutination and precipitation. These are related in that they both involve antibody-antigen reactions that between chemicals with multiple reaction sites. Thus, rather than involving one antibody reacting with one antigen, you get groups of antibodies and antigens reacting together. In the case of precipitation you end up with a chemical precipitate forming that settles out of solution and shows up as a thin white line in the agar. This will form only between corresponding antibodies and antigens.


If the antigen is attached to a cell, such as a RBC, when the antibodies react with them it will form a large clump of chemicals and cells. This is called agglutination and is commonly seen in blood typing reactions.


These types of reactions are being increasingly used to test for the presence of specific pathogens or diseases in a variety of ways. We now have tests for AIDS, Strep throat, rhematoid arthritis and many other diseases available. They can also be used in testing things like meat purity and blood or tissue typing.


The exact nature of the activity today will depend on the availability of these tests but will include a standard blood typing procedure. Remember that the blood type refers to the presence of that type of antigen on the blood cells and the ability of the body to make the opposite type of antibody, as seen in the chart shown in lab.





1. What is serology?


2. What is the relation between the size of the antigen and the ability to form an agglutination reaction?


3. How are these testing sera created?




As you analyze this experiment, make sure you understand the connection between which antibodies and antigens you added and which ones reacted together. Remember that in the blood typing experiment you are adding known antibodies to the unknown blood cells which contain the antigens. Any clumping indicates a positive test for that antigen and means that it is that blood type. If all three agglutinate, it means you have AB+ blood. If only the B factor clumps, it means you have type B- blood.





1. What does it mean if none of the blood spots agglutinate? What kind of blood would you have?


2. Which blood sample did you test? What type of blood did you determine it to be? Where you correct? If not, what might have thrown you off?





Precipitin Tests





As stated in the last module, precipitates are a type of antibody-antigen reaction where a chemical precipitate is formed and drops out of solution. If it occurs in an agar plate, it will be visible as a thin white line between the two sources. We will be using a commercially prepared kit to view several types of precipitin reactions.


In this kit the antigen will be represented by material labeled "Swine Albumin" or albumin from some other animal. This represents a type of blood fraction from that animal that contains antigens which another animal would view as an antigen.


The antibody in this case will be labeled "Anti-swine Albumin in Goat Serum". This is what is called an antiserum as it is serum containing some type of antibody. In this case a goat was given a shot of swine albumin which served as a antigen. The goat's immune system recognized it as such and formed antibodies against it. The goat's serum was then removed and purified into antiserum. This one would react with the antigen mentioned above.


This technique used simple saline agar in which small wells have been dug with a pipette. These wells will be filled with a variety of antibodies, antigens, and controls and allowed to sit for 48 hrs. During this time the reagents will diffuse out into the agar and react if they encounter their corresponding match. If they do not do so, they will not form a precipitin line and you will know that they did not match.


This technique is useful in a number of ways but one test based on this is used to test the purity of meat. If the meat is placed in the center with wells containing antibodies for known meats such as beef, pork, horse, etc. the antigens in the meat will diffuse out and react with the corresponding antibody. This allows the FDA to know if the meat is pure beef or is mixed with something else.


IN lab we will do two tests. One will be a basic precipitate test looking at the effect of antigen concentration on the types of precipitation lines formed. Here, each group will work with a different animal serum. The second test will examine the results of mixing various types of antibodies and antigens to be able to identify similarities and differences between the mixtures. This is fairly complicated and will be explained more in lab with the use of diagrams, photos, etc.


There are other types of antibody-antigen reactions than we have examined in these two modules. Sometimes the two chemicals will react in such a way that the antigen is chemically inactivated. This is called neutralization. Other times the antibody will react with the antigen so that the antigen is marked for easier phagocytosis by a macrophage. This process is called opsonization. If the antigen is on a mobile cell, the antibodies may react with the antigen so that the cell itself is immobilized.





1. Define antiserum.


2. Explain how antiserum against a snake's venom could be made from a horse.


3. List two other types of antibody-antigen reactions besides the two we have worked with here.





The critical point here, obviously, is to find clear precipitate lines. Once these are visible you will need to remember what you put in the two wells that caused such a reaction and what you put into the wells that did not react. Remember to be sure to understand which reagent was an antibody and which was an antigen. Drawing out the petri dish with the wells labeled and the precipitate lines drawn in will help greatly. Notice that the smaller concentrations may actually have given more visible lines.


In the identity reaction test, be sure you understand why the different arrangements of antibodies and antigens cause different shapes of precipitate lines. To do this it would help to use different colors and to diagram out the diffusion patterns that result. Remember that as soon as the antibody encounters a matching antigen, the reaction line is formed and neither reagent diffuses any further. Therefore, for any lines to be formed beyond that point there must be an antibody or antigen that did not react and is thus free to continue to diffuse onward where it can react with a different matching reagent to form a second line. Be sure to redraw the plate you did with the wells clearly labeled with the material they contained and the lines formed shown. This will help you understand what reacted with what and why.





1. In the first test, which concentration gave you the best precipitate line?


2. Create an hypothetical petri dish with five wells. Put an imaginary antibody in the middle and four imaginary but reasonable antigens in the wells around it. Now figure out where precipitate lines would form in this example.


3. Did you see an identity, non-identity or partial identity reaction in your plate for the second test?


4. Diagram what a non-identity reaction line would look like.





Intestinal Pathogens in the Family Enterobacteriaceae





Several of our modules have already brought up the group of gram negative bacilli commonly found in the human intestinal track. While many of these organisms, which are almost impossible to differentiate on the basis of appearance alone, are non-pathogenic, several of the species are significant pathogens. Therefore, it is important to be able to distinguish between the pathogenic ones which may be causing the patient's problems and the non-pathogenic normal flora ones.


The non-pathogenic bacteria would include members of the Escherichia, Proteus, Enterobacter and Pseudomonas genera. The pathogenic strains in this group would be in the Salmonella and Shigella genera. All of these are placed in the Family Enterobacteriaceae.


In order to differentiate these organisms there are several tests that are usually performed, several of which we have done previously. In this exercise we will start with an isolation technique using selective and differential agar and then move on to further identifying tests. Since each gives a different set of reactions to the total group of tests, once completed the organism should be positively identified. Since each step must be done in order this exercise will actually be run over the next week and a half.


The first step is to isolate out the pathogenic organism from the normal flora, as would normally be encountered in a real-life fecal sample. You will provided with a mixed culture composed of one of two combinations of one pathogenic and two non-pathogenic bacteria. Since these contain pathogenic organisms it is extremely important that you follow very strict aseptic techniques.


You will first streak the mixture on both SS agar and MacConkey Agar plates for isolation. Be sure to record which mixture you used! These agars are both designed to select against gram positive bacteria and for gram negatives since they contain bacteriostatic dyes that, as we learned in the gram stain module, inhibit gram positive bacteria. The differential aspect relates to the ability of some of these bacteria to ferment lactose. If they can ferment lactose they will pick it up from the media and, in the process, also pick up the dye. This will cause lactose fermenters to have colored colonies and non-lactose fermenters to have white or clear colonies.


Since the pathogenics are non-lactose fermenters your next step will hopefully make use of any white or clear colonies. You will transfer a pure culture to one of two TSI slants. This technique is unusual as you will first stab through the slant and then you will streak up the slant. This will cause the organism to react with both the base of the media and the surface of the media. Figure 49-1 demonstrates this technique while figure 49-2 shows how to interpret the results. Scion there are several possible combinations of reactions be sure and use this latter figure to interpret what your bacterium does.


To complete this process, you will then transfer inoculum from the TSI slants to the Urea Broths. This is a test that you have already performed and allows you to see if your organism produces urease which allows it to break down urea into carbon dioxide and ammonia. The latter will create an alkaline environment which should turn the pH indicator, phenol red, a deep red or cerise color.


With the results from these last two tests, you should be able to use Table 49-1 to identify which organism you isolated from the original mixture.


While this procedure is fairly accurate, it is time consuming and requires a lot of media, plates, tubes, etc. Doing many of these in a commercial clinical lab would get old very fast. Therefore, several rapid identification systems have been developed to facilitate rapid and efficient identification of enterics. We will use the Enterotube II system in this experiment to demonstrate the way a clinical lab processes these samples. Unfortunately, while this will appear to be a very easy technique, you will soon see why the labs using this method have to train their technicians thoroughly to insure reliable and accurate results.


The technique is easy to do as you simply remove the caps at either end of the unit, dip the inoculating tip into one of the colonies from either the SS or MacConkey plates and, grasping the handle end, pull the tip back and forth through the unit. When this has been done you will need to pull the handle back out slightly and snap it off at the etched location. The broken off end is then used to puncture the sides of the aerobic chambers. Replacing the caps, labeling and placing in the incubator completes the process.


48 hrs. later the unit is removed and compared to the color chart showing before and after illustrations for positive results. Two of the compartments require the addition of chemical reagents. These will be provided in syringes so that the needles can be popped through the thin plastic bottom and the reagents added to each slant. Once results have been recorded on the provided sheet, positive tests are circled, their corresponding values are added up and a five digit code is produced. Comparing this code to a chart will, hopefully, identify the bacterium.





1. What does TSI stand for?


2. List two specific non-pathogenic and two specific pathogenic bacteria discussed in the module. For the pathogenic ones give a disease they cause.


3. Are the normal flora most likely to be lactose fermenters or non-fermenters?


4. What does the term coliform refer to?





While each mixture had one pathogenic species in it, there is a good chance that you will see only colored colonies on the SS and MacConkey plates. These represent the non-pathogenic species which frequently out compete the pathogens in the nice safe environment of the test tube. However, the identification process can occur with the non-pathogenic varieties just as well. Notice that there are probably several types of colonies in the plates representing the several types of bacteria in the original mixture.


The TSI slants are usually easily analyzed unless the production of the H2S and the resultant black precipitate overwhelms the rest of the colors and reactions. The production of gas, in particular, can be quite spectacular with the agar being split and raised significantly in many cases.


The urea hydrolysis is a difficult test to run, as was demonstrated when you performed module 36. This is usually due to the difficulty of insuring sterility given that the media cannot be autoclaved.


Hopefully, the results from these tests will allow you to identify a bacteria that was in your original mixture. Be sure and compare the results you got with what was in your original mixture to see how accurate the process was for you.


The Enterotubes are quite useful once you have been completely trained and have had sufficient experience using them. This is why the companies producing these types of units provide regional two or three-day workshops for the technicians using the products. While it seem simple, the odds of your properly identifying your organism the first time is slim. However, when you get the code and match it to an organism, be sure to check it against what was in your original mixed culture to see if that is one of the possibilities.


Again, it is imperative that you understand the process and the results that each enteric organism can give you. This is one module where you will be expected to memorize the specific results that are critical in separating out this group of organisms.





1. If you had no growth on either of the original streak plates what could it imply?


2. If you ended up with a TSI slant that had a yellow butt, red surface and no gas trapped in it, how would you interpret it?


3. If, after running all the tests you came up with a TSI slant that showed acid and gas from glucose, negative lactose fermentation and the production of H2S, and a positive urease test, which bacterium would you have?


4. Why is it necessary to inoculate the SS plate with two loopfuls in the "O" quadrant?





Identification of Pathogenic Staphylococcus aureus





One of the most serious and most common pathogens for humans is Staphylococcus aureus. A member of the Micrococcaceae family of gram positive cocci, S. aureus is the causal agent of many diseases ranging from mild skin infections to deadly toxic shock syndrome. It is also a common food contaminant causing food poisoning as well as numerous other types of infections throughout the body. It is, therefore, imperative that we have a way to distinguish it from similar organisms.


As we will see in the next two modules, the Streptococcaceae family is also composed of gram positive cocci that are virtually indistinguishable from the Micrococcaceae family. If a near perfect gram stain is done, differentiation may be done in that the Micrococcaceae form clusters while the Streptococcaceae tend to form chains or pairs. However, this is not a very reliable way to separate the two. A better test is to test for the presence of catalase. Catalase is an enzyme that breaks down hydrogen peroxide into oxygen and water, causing the release of bubbles that we usually associate with the cleansing action of H2O2. The Micrococcaceae produce catalase while the Streptococcaceae do not.


Within the Micrococcaceae are a number of bacteria, several of which may be mildly pathogenic. Therefore, it is important to be able to separate S. aureus from these others. As figure 50-1 illustrates, there is a set procedure to separate out these similar bacteria.


The first test usually makes use of the ability to survive high salt (hypertonic) environments. WE can combine this with the organisms ability to ferment a sugar called mannitol by streaking the organisms on Mannitol Salt Agar plates. These contain the sugar, a high concentration of salt and phenol red as a pH indicator. Only the Staphylococcus should grow on this agar as they are salt-tolerant. If the organism is capable of fermenting the mannitol, it should create an acid environment which will turn the media yellow. S. aureus would give this sort of reaction as it can ferment mannitol.


Another fairly reliable test is to streak a blood agar plate with the bacteria. On blood agar, organisms may give one of three results, all based on their ability to produce hemolysin, the enzyme that allows organisms to lyse red blood cells. Alpha hemolysis means that the organism produces a moderate amount of the enzyme so that there is partial clearing around its colony and, perhaps some greening of the area. Beta hemolysis is when the organism produces a large amount of hemolysin, usually indicative of a serious pathogen, and causes a large area of clearing around the colony where the RBC's have been destroyed. Finally, some organisms produce no hemolysin and simply grow without affecting the agar.


The final test is to see if the organisms produce the enzyme coagulase. This enzyme is used by the bacteria cell to produce clotting of the plasma. This can be tested in several way and is usually done by placing some of the culture into some rabbit plasma and waiting to see if it begins to thicken.





1. What color is phenol red at the three pH ranges?


2. What is hemolysis and what does it do?


3. Name four specific diseases caused by S. aureus.


4. What are the four key features of S. aureus that allow us to identify its presence?





Hopefully, you will get good clear results that will enable you to distinguish among the three bacteria tested. However, you may have conflicting results with the S. aureus culture. This is most likely due to a process called attenuation that occurs whenever pathogenic bacteria are kept in pure culture too long. During live inside its normal host, such pathogenic bacteria make use of their pathogenicity to infect the host and survive. If they have a tendency to produce less virulent strains, these are most likely destroyed by the body's defenses. However, those same features that help them to survive in vivo may actually be detrimental to them when they are grown in vitro. Therefore, over time they may lose some of these pathogenic traits and give different than expected results.


Be sure that you compare each result to the chart on pg. 447 and follow the results of the test on the dichotomous key provided.





1. What is attenuation?


2. Where you able to see any differences at all between the three species when viewed in the gram stains?


3. If you had an organism in this group that grew on the salt agar, did not ferment the mannitol, did not produce hemolysin or coagulase, which organism would it be?


4. What diseases S. epidermidis cause?





Pyogenic Streptococci





As mentioned in module 50, the other group of gram positive cocci that contains several serous pathogens are the Streptococcaceace. We will discuss these in the next two modules. AS the discussion states, there are many pathogenic organisms in this family and they cause a number of significant diseases. Be sure to familiarizes yourself with these pathogens and the diseases they cause. Creating a good taxonomic structure for this group has been difficult but Lancefield created a system based on letters ranging from A to O. Each group is characterized in a variety of ways. Fortunately, the majority of streptococci infections are due to those in Lancefield Group A.


Since there are so many potentially pathogenic organisms in this family, being able to distinguish one from the other is very important. Module 51 will deal with those called the Pyogenic Streptococci due to their ability to produce pus at some point in the infection. The primary pathogen in this group is Streptococcus pyogenes, the causal agent for strep throat, scarlet fever and rheumatic heart disease.


Again, we will conduct a series of tests that, when taken together, will provide the means to separate out the various possibilities. We will start with two tests that make use of the blood agar plates mentioned in module 50. In this case, we will also add a disk of bacitracin to the "0" quadrant and look for a zone of inhibition indicating sensitivity to this antibiotic. When taken with the type of growth on the BAP this will allow us to differentiate between several of the bacteria as shown in table 51-1. You will also do a bile esculin hydrolysis test to see which organisms are capable of breaking down the bile esculin and producing a black precipitate. Organisms that can do this are in Group D. Organisms in group D are also capable of surviving hypertonic environments and so they will most likely be the only ones capable of growing in the 6.5% NaCl broth.


A final test will be demonstrated for you due to its complexity. This is the C.A.M.P. test which makes use of the differential growth that occurs when several organisms are grown in a particular pattern on BAP. When Streptococcus pyogenes and Streptococcus agalactiae are grown perpendicular to a streak of Staphylococcus aureus the S. agalactiea will grow in such a way as to create an arrow-shaped pattern near the S. aureus while the S. pyogenes will not.




1. Name three diseases caused by members of Lancefield Group A.


2. What does C.A. M. P. stand for?


3. Name an organism in the Lancefield Groups D and Viridans.


4. Regarding their oxygen requirements, where would most Streptococci fit?





Notice that, once again, it was very difficult to impossible to see a visible difference between the members of the Streptococcaceae on the basis of the gram stain. One can understand the importance of having these types of tests to be able to distinguish between the numerous members of the family since so many are potentially disease causing.


Were you able to see each of the three types of growth on BAP that can result from these species? This is perhaps the best time in the semester to see each of the three patterns at once since the family is capable of producing all three. Remember that most of these organisms are microaerophilic and thus would grow best in a candle jar. That may explain any difficulty you encounter growing them under aerobic conditions.


Remember that with the bacitracin sensitivity test you are basically just doing the Kirby-Bauer method of agar diffusion to teat for antibiotic sensitivity. The only difference is that you are using BAP instead of Mueller-Hinton Agar.


When doing the Bile Esculin test remember that the simple presence of a black precipitate is not sufficient to create a positive reaction. Instead over half of the slant must be blackened for it to be a solid positive result. This test is useful for separating out the Group D bacteria.


Finally, since only the enterococcus streptococci are able to grow in the hypertonic salt broth, only Enterococcus faecalis should grow well in the NaCl broth.





1. Which types of BAP reactions were you able to see? Which organism produced which reaction?


2. Which species were resistant to the effects of bacitracin?


3. Based upon module 50, where do you think you are most likely to encounter the Enterococcus genera?


4. Were you able to distinguish any difference between the two Streptococcus species when grown on the CAMP agar?


5. If you had a Streptococcus species that tested as having beta hemolysis, was sensitive to bacitracin and was negative to the CAMP test, unable to grow in a hypertonic environment or to hydrolyze bile esculin what would it be?





Pneumococci and Other Alpha Streptococci





In Module 51 we dealt with the Streptococcus species that were either beta or gamma hemolytic. In this module we concentrate on those that are typically alpha hemolytic. Again, as shown in the discussion for this module, there are a number of organisms that cause significant human diseases in this group also. Several of these, such as S. pneumoniae, the most frequent cause of bacterial pneumonia, is one of these. It, and the others in this group, do have a different appearance in a well done gram stain. As illustrated in figure 52-1, these organisms frequently grow in capsules as paired cocci. As such they are called diplococci. These diplococci may then appear either alone or in pairs or chains. However, we also have a variety of others tests that are used to separate out these species from each other as illustrated in the dichotomous key shown in figure 52-2.


Just as we used the antibiotic bacitracin to distinguish among the species in module 51, here we will use sensitivity to optochin to help distinguish between two of the more important alpha streptococci. S, mitis will not be affected by this antibiotic while S. pneumoniae will be.


S. pneumoniae is also sensitive to destruction by the common emulsifier called bile. When it is placed in the presence of bile the cells will actually lyse and be destroyed. Thus, when you add bile to a turbid broth of S. pneumoniae it will become clear while the S. mitis will remain cloudy. Care must be taken in performing this test as the pH of the solution prior to adding the bile is critical. Therefore, be sure that it is neutralized before the reagent is added.


Finally, S. pneumoniae is capable of fermenting the sugar inulin (not insulin as is incorrectly written in the dichotomous key). The test done to check this reaction is the same we did with the phenol red broth in the carbohydrate fermentation test we did previously. Here, inulin is added and if it is fermented, the acid produced will cause the phenol red to turn yellow.





1. What does opportunistic mean in the case of a pathogen?


2. What is SBE and what causes it?


3. List three diseases caused by members of this group.


4. How are normal mouth flora frequently introduced into the blood stream?


5. What is the purpose of the bromthymol blue in the bile solubility test?





Again, since S. pneumoniae is a pathogenic organism, this module may be affected by the process of attenuation. Difficulties may also occur because these organisms are microaerophilic and may not grow well in normal aerobic incubators.


AS you examine the plates and tubes in this experiment, be sure to compare them to the dichotomous key in figure 52-2 to see if the results match.


When doing the bile solubility test, notice the change in color when the bromthymol blue is first added to the solution. It should then change back when the NaOH is added indicating that the acid has been neutralized. Once this has been done, the bile can be added via the desocycholate reagent and clearing can be observed. Remember that for this experiment to be successful, you must have turbidity initially due to the growth of the bacteria. If when you remove the tubes from the incubator, there is little or no turbidity, you will not get an accurate test result.


When examining the color change that occurred with the inulin fermentation test be sure to compare your results with module 34 where we previously did this type of carbohydrate fermentation.






1. Were you able to see evidence of the diplococci structure of any of the bacteria?


2. Why do you think an acid environment is produced by the bacteria growing in the Todd Hewitt broths?


3. If you had an organism that gave an alpha hemolysis on BAP, was not affected by the antibiotic optochin or by bile but was not able to ferment the sugar inulin, what organism would you be working with?


4. Diagram a chain of diploccoi.





Dental Caries Susceptibility





As has been seen in lecture, the oral cavity is full of normal microflora that are, usually, living in a commensalistic relationship with us. However, under certain conditions, they may shift and enter a parasitic, disease-causing relationship. This is one of the things that must be kept in mind regarding the organisms that live on and around our teeth.


The majority of these are anaerobic fermenters that are capable of fermenting the sugars we consume and producing the acids that we have encountered in several modules. These acids, then, are held against the enamel of the teeth due to the formation of plaque by the bacteria. The plaque is composed primarily of glucan which is also a result of the fermentation of the sugars we consume. If the plaque holds the lactic acid against the tooth surface too long, then the acid will begin to breakdown the enamel and tooth decay will occur. This will lead to cavities or dental caries.


Several of the principle culprits in this process are listed in the discussion section and you need to be familiar with them.


While this test will give you an idea of the relative number of acid fermenters in your mouth, and thus your possible susceptibility to dental caries, there are numerous other factors that contribute to your overall dental hygiene. The types of foods and drinks you consume, how well you brush and floss, and you genetic history all interact with the number of fermenters to result in your overall likelihood of having cavities. The most important factor, probably, is the amount and quality of brushing as the process of acid and glucan formation takes considerable time. If you continually and regularly disrupt these colonies by brushing and flossing you stand a good chance of reducing their activity.


Since you will be collecting your own sputum be sure to handle it carefully and so as not to contaminate anyone else's work area or material. Be sure that each person handles and disposes of their own bodily fluids properly.





1. What type of agar are we using in this test?


2. How quickly would the acid have to be formed for you to have a marked susceptibility to dental caries?


3. List three species known to produce dental caries.


4. What is the pH indicator used in this experiment?





This test requires that it be checked every 24 hrs. to see exactly when the pH changes occur. Since you are looking simply for a color change, it is fairly simple to interpret. It will either stay the same of change colors indicating that acids have been produced. As soon as it does change color, the experiment may be terminated.




1. How susceptible to dental caries does this test indicate you are?


2. In examining your dental history, do you feel this is an accurate assessment?


3. What could you do to change it?


4. What are the colors that bromcresol green turns in the different pH's?


5. What is unusual about the lactic acid producing bacteria found in the mouth?





Urine Culture





One all too common infection in humans, particularly females, is urinary tract infections caused by a number of bacteria. In this module we will make use of a commercially available product called a DipSlide that is used by clinical labs to rapidily test for such infections.


Usually, this is a three step process as the quantity of bacteria present must be evaluated, then the types of bacteria need to be identified and, finally, the type of antibiotics that it most effective against this strain must be determined. The module activities listed is more complete than we will go into but the simple technique we will use will allow us to do the first two of these steps.


While the upper urinary tract is usually fairly sterile, the opening of the urethra is usually heavily contaminated with microbes, particularly in the female due to its proximity to the rectum and the vagina. If bacteria are able to transverse up the urethra to the bladder or beyond, urinary tract infections will occur.


The types of organisms that are frequently involved in these types of infections are listed in the discussion for the module. Be familiar with them and how they are classified.


The DipSlide is a simple device designed to quickly enumerate the relative amount of bacteria in the urine and to begin the process of identifying the bacteria. It is a two-sided flat stick that has a different type of agar on each side. The type of agar varies from product to product but one will usually be useful for identifying gram positive bacteria while the other will be of use with gram negatives. The urine is first collected and it is best if this is done with proper cleaning and collection to reduce contamination from bacteria surrounding the opening.


Once the urine is collected, the slide is simply dipped into the urine, allowed to drain slightly and then placed into the container. It is important to seal this completely as it can become quite odiferous upon incubation.


After incubation, the slide and its container is removed from the incubator and, without opening the container, compared to the chart provided by the producer of the unit. We will concentrate just on the relative number of bacteria present as in indication of normal flora or infection. However, be sure to look closely at the colonies themselves in light of other things you have learned in this class regarding differential media.


Remember, you will all be handling bodily fluids with the potential of transmitting pathogens through them. Be sure to handle them correctly, to only handle your own and to popery dispose of the urine, the container and the DipSlide itself when each has been used.





1. List two gram positive and two gram negative organisms capable of causing urinary tract infections.


2. How is the Kirby-Bauer method tied in to this lab?


3. What is the most common organism associated with urinary tract infections?


4. What type of media is used to isolate the gram positive bacteria from the urine?


5. Why should the urine be tested as soon as it is collected?





Comparing the growth on your slides against the diagrams provided should allow you to identify if you are currently experiencing a urinary tract infection. However, remember that this is the first time you have done this technique and that we are not doing the collection procedure as properly as we could. Therefore, unless you are experiencing significant symptoms of such an infection, do not be concerned if you show a great deal of contamination.


Besides relative numbers, be sure to examine the difference between the two sides of the slide as they are two different types of media. There may be a difference in quantity, appearance, etc.





1. Did your test results indicate a possible urinary tract infection?


2. Based upon the media used were you primarily identifying gram positive, gram negative or both with the DipSlide you used?


3. What is the correct procedure for collecting urine for this test?


4. What is unusual about Alcaligenes faecalis?





Throat Culture





Due to the continual entrance of air into and out of the oral cavity, along with the food and drink we put in there, the mouth is filled with a wide variety of microorganism. Many of these are considered to be normal flora and exist in commensalistic relationships with us. However, as we have seen in previous modules, a number of these are capable of becoming pathogenic under certain conditions. When this occurs it is necessary to do a throat culture to determine which type of organism is present in numbers sufficient to create a problem.


The discussion section of the module lists numerous examples of pathogenic organisms found in the throat. Be sure to know these and the diseases they may produce.


The process outlined in this module through the various activities is a more complete one that we will be undertaking. We have done many of the tests used in this module already so we will concentrate on a simple initial streak of the throat culture on BAP and MacConkeys and the use of another clinically useful device called a Bullseye Respiratory Plate or a Quad plate. This device contains four different media in one plate and each media is useful for identifying a particular common throat pathogen. By adding the appropriate antibiotic disk we can also test for susceptibility the way we did in identifying the Streptococcus species in modules 51 and 52.


You will work with a partner to take a good throat culture with a sterile cotton swab. The "0" quadrant of two BAP and one MacConkey plate will be inoculated with that swab which will then be disposed of immediately and properly. Another throat swab will be used to inoculate the four sections of the quad plate and the antibiotic disks will be added as instructed. These plates will then be placed in the proper oxygenated environemtn. One BAP and the quad plate will go into microaerophilic environments. The first into a candle jar and the second into special bags that contain a chemical reagent that duplicates the action of the candle. The other BAP plate and the MacConkey plate will be placed into the incubator under aerobic conditions.


After incubation, all plates will be examined for characteristic growth patterns as per your lab manual module 55, previous lab modules and the information provided with the quad plate. Further testing could then be done to more accurately identify the pathogen and to ascertain what antibiotic could be used against it.





1. Name three bacteria that quickly establish themselves after birth.


2. What is the principle causal agent of bacterial meningitis?


3. What does Hemophilus influenza cause?


4. If the throat swab is not to be used immediately or needs to be transported to the clinical lab, how should it be stored?


5. List three specific agars that are used in isolating and identifying throat flora.





In analyzing the BAP plates you need to remember the three types of reactions that are seen on blood agar, what causes each and what they mean in terms of the bacterium causing it. Be sure to also compare the growth and colony size and type of the BAP plate grown in the candle jar and that grown in the incubator under aerobic conditions.


Remember that the MacConkey agar is designed to select for gram negative bacteria and so any growth on that agar will be different organisms than the gram positives that will be favored on the BAP.


When you examine the quad plate be sure to look at the amount of growth on each type of agar and the appearance of each culture on the different agars. By comparing this to the poster provided on the board, and remembering that each agar is designed for a different bacterium, you may be able to tentatively identify the organisms you isolated.





1. Did you have more growth on the microaerophilic BAP or the aerobic BAP?


2. Was their evidence of gram negative bacteria on any of your plates?


3. Did any beta hemolytic cultures show up? How would you know that they did?


4. Did you see any significant differences between the appearance of the throat cultures on the four different types of media in the quad plate? Were you able to identify any particular organisms on the basis of their growth on the quad plate?